Compositions useful in the treatment of CDKL5 deficiency disorder (CDD)

Abstract

A recombinant adeno-associated virus (rAAV) having an AAV capsid and a vector genome comprising a nucleic acid sequence encoding a functional CDKL5 (hCDKLK5) is provided. Also provided is a production system useful for producing the rAAV, a pharmaceutical composition comprising the rAAV, and a method of treating a subject having CDD or ameliorating a symptom or delaying progression of CDD by administering to a subject in need thereof an effective amount of the rAAV.

Description

Background Technology

[0001] CDKL5 deficiency (CDD) is a serious neurodevelopmental disorder affecting young children. The underlying cause is a mutation in the X-linked cyclin-dependent kinase-like 5 gene CDKL5 (Mendelian Inheritance in Man, MIM:300203; formerly known as STK9), leading to a lack of CDKL5 protein expression and resulting in a range of phenotypes, including EIEE2 (MIM:300672), a form of early infantile epileptic encephalopathy [Bahi-Buisson, N. et al. Key clinical features to identify girls with CDKL5 mutations. Brain 131, 2647-2661, doi:10.1093 / brain / awn197 (2008)] and infantile spasms [Fehr, S. et al. European Journal of Human Genetics (Eur J Hum Genet) 21,266-273, doi:10.1038 / ejhg.2012.156(2013); Kalscheuer, VM et al., American Journal of Human Genetics, 72,1401-1411, doi:10.1086 / 375538(2003); Tao, J. et al., American Journal of Human Genetics, 75,1149-1154, doi:10.1086 / 426460(2004); Weaving, LS et al., American Journal of Human Genetics, 75,1079-1093, doi:10.1086 / 426462(2004)). In addition to the characteristic early-onset seizures, the phenotype may also include several other features, such as stereotyped hand movements, severe psychomotor retardation, and generalized hypotonia. The onset of early postnatal symptoms suggests that CDKL5 plays a crucial role in brain development. CDKL5 is also expressed in the mature adult nervous system. CDKL5 is expressed throughout the cell, including the cell body, dendrites, nucleus, and cytoplasm.

[0002] Mutations in the CDKL5 gene are the cause of most cases of CDD (a progressive neurodevelopmental disorder) and are one of the most common causes of cognitive impairment in women. Men with gene mutations that cause CDD are devastated. Most of them die before birth or in early infancy. See, for example, ninds.nih.gov / Disorders / Patient-Caregiver-Education / Fact-Sheets / Rett-Syndrome-Fact-Sheet and omim.org / entry / 312750.

[0003] Currently, there is no cure for CDD, and treatment focuses on alleviating the symptoms. In many cases, seizures are difficult to control, therefore there is an urgent medical need to find new treatment methods. Summary of the Invention

[0004] This article provides a recombinant adeno-associated virus (rAAV) for the treatment of CDKL5 deficiency (CDD) in subjects of need. The rAAV carries a vector genome comprising an inverted terminal repeat (ITR) sequence and a novel nucleic acid sequence encoding a functional hCDKL5 protein under the control of regulatory sequences that guide the expression of human CDKL5 in target cells.

[0005] In some embodiments, a recombinant adeno-associated virus (rAAV) for treating CDD is provided. The rAAV comprises: (a) an AAV capsid; and (b) a vector genome packaged within the AAV capsid of (a), wherein the vector genome comprises an inverted terminal repeat (ITR) sequence and a nucleic acid sequence encoding a functional human CDKL5 (hCDKLK5), the nucleic acid sequence being under the control of a regulatory sequence directing the expression of hCDKL5 in central nervous system cells, and wherein the hCDKL5 encoding sequence is nt 699 to 3581 of SEQ ID NO:3 (or SEQ ID NO:22) or is at least about 95% identical to nt 699 to 3581 of SEQ ID NO:3 (or SEQ ID NO:22). In some embodiments, the functional hCDKL5 has the amino acid sequence of SEQ ID NO:2. In some embodiments, the regulatory sequence comprises a ubiquitin C (UbC) promoter. In some embodiments, the regulatory sequence comprises a WPRE element.

[0006] In some embodiments, a recombinant adeno-associated virus (rAAV) for treating CDD is provided, wherein the functional human CDKL5 (hCDKLK5) is hCDKL5 isotype 2. In some embodiments, a recombinant adeno-associated virus (rAAV) for treating CDD is provided, wherein the functional human CDKL5 (hCDKLK5) is hCDKL5 isotype 3. In some embodiments, a recombinant adeno-associated virus (rAAV) for treating CDD is provided, wherein the functional human CDKL5 (hCDKLK5) is hCDKL5 isotype 4.

[0007] In some embodiments, the rAAV used to treat CDD includes an AAV9 capsid. In some embodiments, the rAAV used to treat CDD includes an AAVhu68 capsid. In other embodiments, the rAAV used to treat CDD includes an AAVrh91 capsid.

[0008] In some embodiments, the CDKL5-2GS encoding sequence is SEQ ID NO:24 or a sequence that is at least 95% identical to SEQ ID NO:6. In some embodiments, the CDKL5-3GS encoding sequence is SEQ ID NO:25 or a sequence that is at least 95% identical to SEQ ID NO:8. In some embodiments, the CDKL5-4GS encoding sequence is SEQ ID NO:26 or a sequence that is at least 95% identical to SEQ ID NO:10.

[0009] In some embodiments, the vector genome includes at least two tandem repeat sequences of a dorsal root ganglion (drg) specific miRNA target sequence in the 3' untranslated region of hCDKL5, wherein the at least two tandem repeat sequences include at least one first miRNA target sequence and at least one second miRNA target sequence, which may be the same or different, and target miR183 or miR182.

[0010] In some embodiments, vectors other than rAAV are used to deliver expression cassettes as described herein. The vectors may be recombinant parvovirus, recombinant lentivirus, recombinant retrovirus, or recombinant adenovirus; or nonviral vectors selected from naked DNA, naked RNA, inorganic particles, lipid particles, polymer-based vectors, or chitosan-based formulations.

[0011] In some embodiments, a composition is provided comprising a stock solution of rAAV or other viral vectors as described herein and an aqueous suspension medium.

[0012] In some embodiments, a method for treating CDD is provided, the method comprising administering an effective amount of the rAAV or other carrier described herein to a subject in need.

[0013] In some embodiments, an rAAV generation system is provided that can be used to generate vectors as described herein.

[0014] In some embodiments, a vector is provided comprising an expression cassette, wherein the expression cassette includes a nucleic acid sequence encoding a functional human CDKL5-co1 (hCDKL5), the nucleic acid sequence being under the control of a regulatory sequence directing hCDKL5 expression, and wherein the hCDKL5 encoding sequence is SEQ ID NO:22 or a sequence at least about 95% identical to SEQ ID NO:22. In some embodiments, a vector is provided comprising an expression cassette, wherein the expression cassette includes a nucleic acid sequence encoding a functional human CDKL5-2GS (hCDKL5-2GS), the nucleic acid sequence being under the control of a regulatory sequence directing hCDKL5-2GS expression, and wherein the hCDKL5-2GS encoding sequence is SEQ ID NO:24 or a sequence at least about 95% identical to SEQ ID NO:24. In some embodiments, a vector is provided comprising an expression cassette, wherein the expression cassette includes a nucleic acid sequence encoding a functional human CDKL5-3GS (hCDKL5-3GS), the nucleic acid sequence being under the control of a regulatory sequence directing hCDKL5-3GS expression, and wherein the hCDKL5-3GS encoding sequence is SEQ ID NO:25 or a sequence at least about 95% identical to SEQ ID NO:25. In some embodiments, a vector is provided comprising an expression cassette, wherein the expression cassette includes a nucleic acid sequence encoding a functional human CDKL5-4GS (hCDKL5-4GS), the nucleic acid sequence being under the control of a regulatory sequence directing hCDKL5-4GS expression, and wherein the hCDKL5-4GS encoding sequence is SEQ ID NO:26 or a sequence at least about 95% identical to SEQ ID NO:26.

[0015] In some embodiments, the vector genome further includes at least two tandem repeat sequences of a dorsal root ganglion (drg)-specific miRNA target sequence. In some embodiments, the rAAV or a composition including the rAAV may be administered to subjects in need to improve symptoms of CDD and / or delay the progression of CDD.

[0016] In another aspect, this document provides a composition comprising rAAV or a carrier as described herein and an aqueous suspension medium.

[0017] On the other hand, a method is provided for treating a subject with CDD, improving the symptoms of CDD, or delaying the progression of CDD. The method includes administering an effective amount of rAAV or a carrier as described herein to the subject in need. In some embodiments, the carrier or rAAV may be administered to the patient via intracerebellomedullary cistern injection (ICM).

[0018] These and other aspects of the invention will become apparent from the following detailed description of the invention. Attached Figure Description

[0019] Figure 1A An AAV vector design for an AAV CDKL5 vector genome and expression cassette is shown. The AAV CDKL5 vector genome includes a 5' AAV inverted terminal repeat (ITR), and the expression cassette includes a neuronal promoter, an engineered human CDKL5 DNA coding sequence, an enhancer and polyA, and an AAV 3' ITR. Figure 1B An AAV vector design for an AAV CDKL5 vector genome and expression cassette is shown, wherein the AAV CDKL5 vector genome includes a 5' AAV ITR and the expression cassette includes a ubiquitin C (UbC) promoter, an engineered human CDKL5 DNA coding sequence, a drg-off-target miRNA, polyA, and an AAV 3' ITR. Figure 1C An AAV vector design for an AAV CDKL5 vector genome and expression cassette is shown, wherein the AAV CDKL5 vector genome includes a 5' AAV ITR and the expression cassette includes: a chicken β-actin heterozygous promoter (CBh), an engineered human CDKL5 DNA coding sequence, drg-off-target miRNA, polyA, and AAV 3'ITR.

[0020] Figure 2A and 2B Analysis of the mouse hippocampus as assessed using anti-CDKL5 antibody (S957D, University of Dundee, UK) is shown. The hippocampus was administered via neonatal lateral ventricle injection of 5 × 10⁻⁶ antibodies. 10 Mice were treated with GC AAV-hSyn-CDKL5-1co.WPRE. Figure 2A The CDKL5 / tubulin levels in wild-type mice injected with PBS, KO mice injected with PBS, and treated KO mice were plotted by Western blot analysis of CDKL5 expression. Figure 2B The CDKL5 activity is shown based on the pS222EB2 (Baltussen et al., 2018) level in wild-type mice injected with PBS, KO mice injected with PBS, and treated KO mice.

[0021] Figure 3 Figures are provided showing the maturation and survival of mice in a CDD mouse model after injection of AAV-hSyn-hCDKL5-1co.WPRE. All injected mice survived and gained weight after treatment. These mice did not show any obvious signs of adverse consequences.

[0022] Figure 4 Behavioral assessment results of CDD mice treated with AAV-hSyn-CDKL5-1co.WPRE are presented. This chart is from the Elevated Zero Maze, which assesses the balance between adventure, curiosity, and anxiety. The first bar represents wt mice treated with PBS. wt mice are curious but cautious and spend limited time outdoors. The middle bar shows Cdkl5-ko mice treated with PBS only, which showed reduced anxiety and spent more time outdoors, entering open areas more frequently. After AAV-CDKL5 treatment, the behavior of Cdkl5-ko mice reverted to wt behavior (in terms of time spent in open areas and number of times entering open areas from closed areas).

[0023] Figure 5A and 5B Behavioral assessment results of CDD mice receiving AAV-hSyn-CDKL5-1co.WPRE are provided. Figure 5A The activity of mice exploring an open arena is shown and plotted as beam interruption times / box-to-box time (min). Dashed lines represent wt mice, which were curious but explored the arena within 10 minutes and then calmed down. Lines with long horizontal bars represent Cdkl5-ko mice that spent more time exploring but eventually calmed down. Lines with horizontal bars and dots represent Cdkl5-ko mice whose activity decreased after AAV-CDKL5 treatment, and whose overall activity level was similar to that of wt mice. Figure 5B The cumulative activity data of the total number of beam interruptions obtained by the mice are shown, which confirms... Figure 5A The results in.

[0024] Figure 6 The graph shows the fall delay time (seconds) measured over three consecutive days in the motor activity and agility assessment (rotand) of mice. Over time, increased performance was observed in wild-type mice during learning. Cdkl5-ko mice exhibited improved performance compared to wt mice, likely due to the previously observed initial hyperactivity. After treatment with AAV-hSyn-CDKL5-1co.WPRE, the behavior of Cdkl5-ko mice did not revert to wt behavior.

[0025] Figure 7A and 7B The results of hippocampal learning and memory (Y maze) are shown. Figure 7A The percentage of spontaneous changes is shown in the test group and the two control groups. Figure 7B The distances (m) traveled by the test group and two control groups are shown. The wt mice exhibited a strong tendency to explore maze arms that the mice had not recently visited (spontaneous behavioral alteration).

[0026] Figures 8A to 8D The expression or activity levels of CDKL5 in AAV.CDLK5 vector constructs expressing isotype 1, isotype 2, isotype 3, or isotype 4 were demonstrated. Figure 8A The results show that, compared with wild-type mice injected with the mediated agent and knockout mice injected with the mediated agent, mice injected with AAV vector (5 × 10⁻⁶) showed significant differences. 10 Quantitative expression levels of CDKL5 isotypes 1, 2, 3 and 4 in GC (neonatal ICV) knockout mice. Figure 8B CDKL5 activity was demonstrated by Western blot analysis of tissues from treated wild-type mice (injected with medulant (PBS)) and knockout mice (injected with medulant or AAV.CDKL5-1co), as determined by quantitative signals from pS222EB2. Figure 8C This demonstrates the effects of treatment on wild-type mice (injected with mediated (PBS)) and knockout mice (injected with mediated or AAV.CDKL5-isotypes 1, 2, 3 or 4) derived from PBS. Figure 8A Western blot analysis of tissues, such as CDKL5 activity determined by the quantitative signal of pS222EB2. Figure 8D This shows the quantitative CDKL5 expression level of isotype 1 in KO mice (from... Figure 8B ).

[0027] Figures 9A to 9F This demonstrates the effects of AAV.CDKL5 gene therapy on different vector doses (5 × 10⁻⁶). 10 GC, 2.5×10 10 GC, 1×10 10 GC and 6×10 9 The therapeutic efficacy in knockout mice and wild-type mice was compared in mouse studies using GC. Figure 9A The dosage of 5×10 was shown. 10 Mice treated with GC-AAV.CDKL5 or PBS gained weight (g) over a 10-week period. Figure 9B The dosage of 2.5 × 10⁻⁶ was shown. 10 Mice treated with GC-AAV.CDKL5 or PBS showed a weight gain (g) over a 10-week period. Figure 9CThe AAV.CDKL5 treatment group showed improvement compared to untreated Cdkl5-ko mice at 5 × 10⁻⁶ mm. 10 Dose-dependent results of hind limb clenching tests at GC doses. Figure 9D The results showed that, compared with untreated Cdkl5-ko mice, the AAV.CDKL5-treated group had lower activity levels at 2.5 × 10⁻⁶. 10 Dose-dependent results of hind limb clenching tests at GC doses. Figure 9E The AAV.CDKL5 treatment group showed improvement compared to untreated Cdkl5-ko mice at 1×10⁻⁶. 10 Dose-dependent results of hind limb clenching tests at GC doses. Figure 9F The AAV.CDKL5-treated group showed improvement compared to untreated Cdkl5-ko mice at 6 × 10⁶ mm / kg / year. 9 Dose-dependent results of the hind limb buckling test at GC dosage. WT mice did not exhibit buckling, while KO mice showed significant buckling. The number of bucklings in KO mice was significantly reduced after treatment. WT mice were unaffected by GC injection.

[0028] Figures 10A to 10E This study demonstrates the therapeutic efficacy of AAV.CDL5 gene therapy in CDD ko mice. Figure 10A The AAV.CDKL5 treatment group showed improvement compared to untreated Cdkl5-ko mice at 5 × 10⁻⁶ mm. 10 Nesting results (nest quality / score) at GC dosage. Figure 10B The results showed that compared with WT mice and Cdkl5-ko mice, at 5×10 10 Results of bead embedding tasks with normalization tendency in the AAV.CDLK5 treatment group at GC doses. Figure 10C The results showed that, compared with untreated Cdkl5-ko mice, the AAV.CDKL5-treated group had lower activity levels at 2.5 × 10⁻⁶. 10 Nesting results (nest quality / score) at GC dosage. Figure 10D The results showed that compared with WT mice and Cdkl5-ko mice, at 2.5 × 10⁻⁶... 10 Results of bead embedding tasks with normalization tendency in the AAV.CDLK5 treatment group at GC doses. Figure 10E The AAV.CDKL5 treatment group showed improvement compared to untreated Cdkl5-ko mice at 1×10⁻⁶. 10 Nesting results (nest quality / score) at GC dosage.

[0029] Figures 11A to 11F This demonstrates the correction of hyperactivity in ko mice treated with AAV.CDKL5, as assessed in open field activity tests. Figure 11AThe results show that AAV.CDKL5-treated Ko mice at 5 × 10 10 Walking activity / box-to-box time at GC doses (5-minute intervals to 30-minute intervals). Figure 11B The results show that AAV.CDKL5-treated Ko mice at 5 × 10 10 All activities performed at GC dosage. Figure 11C The results show that AAV.CDKL5-treated Ko mice at 2.5 × 10⁻⁶ mmol / L were used. 10 Walking activity / box-to-box time at GC doses (5-minute intervals to 30-minute intervals). Figure 11D The results show that AAV.CDKL5-treated Ko mice at 2.5 × 10⁻⁶ mmol / L were used. 10 All activities at GC dosage. Figure 11E The results show that AAV.CDKL5-treated Ko mice at 6 × 10 9 Walking activity / box-to-box time at GC doses (5-minute intervals to 30-minute intervals). Figure 11F The results show that AAV.CDKL5-treated Ko mice at 6 × 10 9 All activities at GC dosage. Normalized risk was observed in the elevated zero maze, and normalized hippocampal learning deficits were observed in the Y maze of AAV.CDKL5-treated Ko mice.

[0030] Figure 12 This demonstrates the effect of 5x10 in ko mice. 10 When evaluated at GC doses, expression of CDKL5 isotypes 2–4 provided significant correction for the hindlimb buckling phenotype.

[0031] Figures 13A to 13D The study demonstrated a strong correction trend in KO mice treated with AAV.CDKL5-isotype 1. Figure 13A It is shown in 5×10 10 KO mice treated with AAV.CDKL5-isotype 1 at GC doses showed increased activity. Figure 13B It is shown at 2.5×10 10 KO mice treated with AAV.CDKL5-isotype 1 at GC doses showed increased activity. Figure 13C It is shown in 5×10 10 Activity in the Y maze in KO mice treated with AAV.CDKL5-isotype 1 at GC dose. Figure 13D It is shown at 2.5×10 10 Activity in the Y maze in KO mice treated with AAV.CDKL5-isotype 1 at GC dose.

[0032] Figures 14A to 14CThe results show sex-specific results of hindlimb tethering after treatment of knockout mice with AAV.CDKL5-isotype 1. Figure 14A The hind limbs of male knockout mice treated with AAV.CDKL5-isotype 1 are shown fastened. Figure 14B Hind limb tethering was observed in female knockout mice treated with AAV.CDKL5-isotype 1. In Cdkl5-ko mice, both hemizygous males and heterozygous females exhibited hind limb tethering, which significantly decreased after treatment. None of the WT group showed tethering. Figure 14C It was shown that at high doses (5×10) 10 Walking activity was significantly improved in female heterozygous mice under GC (newborn ICV) conditions.

[0033] Figure 15A and 15B The results show gender-specific findings of open field-walking activity in open field assessment. Figure 15A The results of walking activity in open field assessments of male (KO) mice treated with AAV.CDKL5-isotype 1 are shown, plotted as X-axis and Y-axis beam blockages over time. Figure 15B Results of walking activity in open field assessments of female (ht) mice treated with AAV.CDKL5-isotype 1 are shown, plotted as X-axis and Y-axis beam blockage counts over time. Complete correction to wild-type behavior was observed at multiple time points in both male (KO) and female (ht) mice treated with AAV.CDKL5-isotype 1.

[0034] Figure 16A and 16B Sex differences in ko mice treated with AAV.CDKL5-isotype 1 vector are shown. Figure 16A The results of open field-walking activity in an elevated zero maze assessment of male (KO) mice treated with AAV.CDKL5-isotype 1 are shown, plotted as time (seconds) spent in the open area. Figure 16B Results of open-field walking activity in an elevated zero maze assessment are shown for female (ht) mice treated with AAV.CDKL5-isotype 1, plotted as time (seconds) spent in the open area. Risk-prone behaviors were corrected for, with body size effects being more pronounced in males.

[0035] Figure 17 The distribution of the vector in various tissue samples from NHP studies is provided (in 1×10⁻⁶). 14(GC dose). The figure shows rAAV.CDKL5 in the GC / diploid genome of various non-neuronal tissues, spinal tracts, and brain tissues. Strong transduction in the dorsal root ganglion (DRG) was observed. Moderate to low transduction in brain tissues was observed, with some leakage into non-neuronal tissues.

[0036] Figure 18 Results of quantified hCDKL5 expression (measured by RT-qPCR) in NHP studies, shown in the cerebellum, frontal cortex, occipital cortex, parietal cortex, and temporal cortex, are provided.

[0037] Figure 19A and 19B The results of a dose-escalation study are shown, which measured behavioral changes in WT mice following administration of CDKL5 gene therapy. Figure 19A The results show that, compared to control mice treated with PBS, mice injected with 7.5 × 10⁻⁶ PBS... 10 GC and 1×10 11 In GC AAV WT mice, there was no significant change in the severity score of hind limb tethering. Figure 19B The results show that, compared to control mice treated with PBS, mice injected with 7.5 × 10⁻⁶ PBS... 10 GC and 1×10 11 In GC AAV-treated WT mice, there were no significant changes in walking activity. Detailed Implementation

[0038] This article provides compositions and methods for treating CDD. An effective amount of recombinant adeno-associated virus (rAAV) having an AAV capsid (e.g., AAVhu68 or AAV-PHP.B) and a vector genome packaged within the AAV capsid is delivered to a subject in need, the vector genome encoding a functional human cyclin-dependent kinase-like 5 (hCDKL5).

[0039] I. CDKL5

[0040] The cyclin-dependent kinase-like 5 (CDKL5, also known as CFAP247, serine / threonine kinase 9, STK9; Uniprot#076039) gene is naturally located on the short (p) arm of chromosome X at position 22.13. The N-terminus of the CDLK5 protein acts as a kinase, an enzyme that alters the activity of other proteins. Several direct substrates of CDKL5 have been identified (Baltussen et al., 2018; Munoz et al., 2018). The function of the C-terminus of CDKL5 is unknown.

[0041] As used herein, a functional hCDKL5 protein refers to an isotype, native variant, variant, polymorph, or truncated form of the CKDL5 protein that is independent of CDD and / or whose delivery or expression can improve symptoms of CDD or delay the progression of CDD in animal models or patients. See OMIM#300203, each of which is incorporated herein by reference in its entirety. In some embodiments, a functional hCDKL5 has the amino acid sequence of SEQ ID NO:2 (isotype 1) or an amino acid sequence that is at least about 90% (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to it. In some embodiments, the functional hCDKL5 protein has the amino acid sequence of SEQ ID NO:19 (isotype 2) or at least about 90% (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to it. In some embodiments, the functional hCDKL5 protein has the amino acid sequence of SEQ ID NO:20 (isotype 3) or at least about 90% (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to it. In some embodiments, the functional hCDKL5 protein has the amino acid sequence of SEQ ID NO:21 (isotype 4) or an amino acid sequence that is at least about 90% (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to it. In some embodiments, the functional hCDKL5 is a truncated hCDKL5 comprising a methyl-CpG binding domain (MBD) and an NCoR / SMRT interaction domain (NID) having the aforementioned sequence. See WO2018172795A1, which is incorporated herein by reference in its entirety.

[0042] In some embodiments, functional hCDKL5 protein has improved symptoms of CDD or delayed its progression in animal models. An illustrative animal model is the CDKL5-ko mouse. Other suitable models are described herein.

[0043] Various assays / methods can be used to assess CDD symptoms or progression, including, but not limited to, survival curves (e.g., the Kaplan-Meier survival plot), monitoring body weight, and observing behavioral changes (e.g., by hindlimb clasping, open field assay (motor function), elevated maze (anxiety / risk-to-exploration), Y-maze (learning and memory / hippocampus), bead embedding assay (innate behavior and movement), nesting (innate social behavior), and rotarod assay (motor function, coordination)). In some embodiments, administration or expression of functional hCDKL5 protein in animal models results in improvement of CDD symptoms or delay in CDD progression, said improvement or said delay being demonstrated by assay results being at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% of the assay results obtained in the corresponding wild-type animals. In some embodiments, administration or expression of the functional hCDKL5 protein in CDD animal models resulted in improvement of CDD symptoms or delay in CDD progression, the improvement or delay being demonstrated by improved assay results, which were at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% of the assay results obtained from the corresponding untreated CDD animals.

[0044] This document provides a nucleic acid sequence encoding a functional hCDKL5 protein, referred to as the hCDKL5 coding sequence or CDKL5 coding sequence. In some embodiments, the hCDKL5 coding sequence is SEQ ID NO:3 or a sequence that is at least about 95% identical to SEQ ID NO:3. In some embodiments, the hCDKL5 encoding sequence is selected from SEQ ID NO:2 (referred to as CDKL5 or CDKL5co or CDKL5-1 or CDKL5-1co) or the NCBI reference sequence NM_001037343.1 (referred to as CDKL5 or CDKL5e1; SEQ ID NO:16) encoding the amino acid sequence NP_001032420.1 (SEQ ID NO:19); NM_001323289.2 (SEQ ID NO:17) encoding the amino acid sequence NP_001310218.1 (SEQ ID NO:20); and NM_003159.2 (SEQ ID NO:21) encoding the amino acid sequence NP_003150.1 (SEQ ID NO:21). NO:18); or a nucleic acid sequence that is at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to it. Each NCBI reference sequence in the NCBI reference sequences is incorporated herein by reference in its entirety. In some embodiments, the hCDKL5 coding sequence is modified or engineered (hCDKL5 or hCDKL5co or CDKL5-1 or CDKL5-1co). The modified or engineered coding sequence has less than about 70% (e.g., about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identity with the NCBI reference sequence.

[0045] In some embodiments, the hCDKL5 coding sequence is SEQ ID NO:22 or a nucleic acid sequence that is at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9%) identical to it. In some embodiments, the hCDKL5 coding sequence is SEQ ID NO:24 or a nucleic acid sequence that is at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9%) identical to it. In some embodiments, the hCDKL5 coding sequence is SEQ ID NO:25 or a nucleic acid sequence that is at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9%) identical to it. In some embodiments, the hCDKL5 coding sequence is SEQ ID NO:26 or a nucleic acid sequence that is at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9%) identical to it.

[0046] In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:37 or a sequence that is at least about 95% identical to SEQ ID NO:37. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:38 or a sequence that is at least about 95% identical to SEQ ID NO:38. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:39 or a sequence that is at least about 95% identical to SEQ ID NO:39. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:40 or a sequence that is at least about 95% identical to SEQ ID NO:40. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:41 or a sequence that is at least about 95% identical to SEQ ID NO:41. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:42 or a sequence that is at least about 95% identical to SEQ ID NO:42. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:43 or a sequence that is at least about 95% identical to SEQ ID NO:43. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:44 or a sequence that is at least about 95% identical to SEQ ID NO:44. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:45 or a sequence that is at least about 95% identical to SEQ ID NO:45. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:46 or a sequence that is at least about 95% identical to SEQ ID NO:46. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:47 or a sequence that is at least about 95% identical to SEQ ID NO:47. In some embodiments, the hCDKL5 encoding sequence is an engineered sequence of SEQ ID NO:48 or a sequence that is at least about 95% identical to SEQ ID NO:48.

[0047] As described herein, “nucleic acid” can be RNA, DNA, or a modification thereof, and can be single-stranded or double-stranded, and can be selected from, for example, the group consisting of: nucleic acids encoding proteins of interest, oligonucleotides, nucleic acid analogs such as peptide-nucleic acids (PNAs), pseudo-complementary PNAs (pc-PNAs), locked nucleic acids (LNAs), etc. Such nucleic acid sequences include, for example, but not limited to, nucleic acid sequences encoding proteins, for example, those acting as transcriptional repressors, antisense molecules, ribozymes, small repressive nucleic acid sequences such as, for example, but not limited to, RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense oligonucleotides, etc.

[0048] In the context of nucleic acid sequences, the terms "percentage (%) identity," "sequence identity," "percent sequence identity," or "percent identical" refer to the fact that residues in two sequences are identical when compared to obtain correspondence. The length of a sequence identity comparison is expected to exceed the full length of the genome, the full length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides. However, identity between smaller fragments may also be expected, for example, at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, or at least about 36 or more nucleotides.

[0049] The percentage identity of the amino acid sequence on the full-length protein, polypeptide, approximately 32 amino acids, approximately 330 amino acids, or peptide fragments thereof, or the corresponding nucleic acid sequence coding sequence, can be easily determined. Suitable amino acid fragments can be at least approximately 8 amino acids in length and can be up to approximately 700 amino acids. Typically, when referring to “identity,” “homology,” or “similarity” between two different sequences, “alignment” sequences are used to determine “identity,” “homology,” or “similarity.” An “alignment” sequence or “alignment” refers to multiple nucleic acid or protein (amino acid) sequences, typically containing corrections for missing or additional bases or amino acids, compared to a reference sequence.

[0050] Alignment is performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, such as “Clustal X,” “Clustal Omega,” “MAP,” “PIMA,” “MSA,” “BLOCKMAKER,” “MEME,” and the “Match-Box” program. Typically, any of these programs are used with default settings, although those skilled in the art may change these settings as needed. Alternatively, those skilled in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as provided by the reference algorithm and program. See, for example, JDthomson et al., Nucleic Acids Res., “A comprehensive comparison of multiple sequence alignments,” 27(13):2682-2690 (1999).

[0051] Multiple sequence alignment programs can also be used for nucleic acid sequences. Examples of such programs include "Clustal W", "Clustal Omega", "CAP Sequence Assembly", "BLAST", "MAP", and "MEME", which are accessible via web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the vector NTI utility is also used. Many algorithms known in the art can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, the Fasta program of GCG version 6.1 can be used. TM Comparing polynucleotide sequences. Fasta TM It provides the alignment of the best overlap region between the query sequence and the search sequence, as well as the percentage of sequence identity. For example, the percentage of sequence identity between nucleic acid sequences can be calculated using FastAnalog, as provided in GCG version 6.1, with its default parameters (font size 6 and NOPAM coefficients of the scoring matrix). TM The procedure is hereby incorporated herein by reference.

[0052] II. Expression Box

[0053] This document provides nucleic acid sequences, also known as expression cassettes, that include an hCDKL5 coding sequence under the control of a regulatory sequence that directs hCDKL5 expression in target cells. As used herein, an "expression cassette" refers to a nucleic acid molecule that includes a coding sequence (e.g., a CDKL5 coding sequence) and a regulatory sequence operatively linked thereto. In some embodiments, the vector genome contains two or more expression cassettes. The term "transgenic" refers to a foreign DNA sequence inserted into a target cell; typically, a transgenic encodes a product (e.g., CDKL5). Typically, such expression cassettes loaded into viral vectors contain the coding sequence of the gene product described herein, flanked by packaging signals and other expression control sequences of the viral genome, as described herein. The desired regulatory sequence is operatively linked to the coding sequence in a manner that allows the hCDKL5 coding sequence to be transcribed, translated, and / or expressed in target cells. As used herein, an "operatively linked" sequence includes a sequence adjacent to the hCDKL5 coding sequence that regulates transcription, translation, and / or expression, and a regulatory sequence that acts trans- or distally to control the hCDKL5 coding sequence. The expression cassette may contain a regulatory sequence upstream (5') of the gene sequence, such as one or more promoters, enhancers, introns, etc., and one or more enhancers, or a regulatory sequence downstream (3') of the gene sequence, such as a 3' untranslated region (3'UTR) including a polyadenylation site, and other elements. Such regulatory sequences typically include one or more of, for example, promoters, enhancers, introns, Kozak sequences, polyadenylation sequences, and TATA signals. In some embodiments, the promoter is a tissue-specific promoter, such as a CNS-specific or neuron-specific promoter. In some embodiments, the promoter is the human synaptic protein promoter (SEQ ID NO:23). In some embodiments, additional or alternative neuron-specific promoter sequences may be selected from neuron-specific enolase (NSE) promoters (Andersen et al., (1993) Cell.Mol.Neurobiol., 13:503-15), neurofilament light chain gene promoters (Piccioli et al., (1991) Proceedings of the National Academy of Sciences of the United States of America, 88:5611-5), neuron-specific vgf gene promoters (Piccioli et al., (1995) Neuron, 15:373-84) and / or others.

[0054] In some embodiments, the human synaptic protein promoter has a sequence (e.g., nt213 to nt678 of SEQ ID NO:1, 3, 5, 7, 9 or SEQ ID NO:23), also referred to herein as hSyn or Syn.

[0055] In other embodiments, the promoter is a constitutive promoter, such as the chicken β-actin promoter with the cytomegalovirus enhancer (CB7) promoter, the human elongation initiation factor 1α promoter (EF1a) promoter, or the human ubiquitin C (UbC) promoter. In some embodiments, the regulatory sequence directs hCDKL5 expression in central nervous system (CNS) cells.

[0056] In some embodiments, the target cells may be central nervous system cells. In some embodiments, the target cells are one or more of excitatory neurons, inhibitory neurons, glial cells, cortical cells, prefrontal cortex cells, cerebral cortex cells, and spinal cord cells. In some embodiments, the target cells are peripheral nervous system (PNS) cells, such as retinal cells. Cells other than those from the nervous system may also be selected as target cells, such as monocytes, B lymphocytes, T lymphocytes, NK cells, lymph node cells, tonsil cells, bone marrow mesenchymal cells, stem cells, bone marrow stem cells, heart cells, epithelial cells, esophageal cells, gastric cells, fetal cut cells, colon cells, rectal cells, liver cells, kidney cells, lung cells, salivary gland cells, thyroid cells, adrenal cells, breast cells, pancreatic cells, islet cells, gallbladder cells, prostate cells, bladder cells, skin cells, uterine cells, cervical cells, testicular cells, or any other cells expressing functional CDKL5 protein in a subject without CDD.

[0057] In some embodiments, additional or alternative promoter sequences may be included as part of the expression control sequence (regulatory sequence), for example, located between a selected 5' ITR sequence and the coding sequence. Constitutive promoters, regulated promoters [see, for example, WO 2011 / 126808 and WO 2013 / 04943], tissue-specific promoters, or promoters that respond to physiological cues can be utilized in the vectors described herein. One or more promoters may be selected from different sources, such as the immediate early enhancer / promoter of human cytomegalovirus (CMV), the early enhancer / promoter of SV40, the JC polyomavirus promoter, the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, the latency-associated promoter (LAP) of herpes simplex virus (HSV-1), the long terminal repeat (LTR) promoter of Raoul's sarcoma virus (RSV), the neuron-specific promoter (NSE), the platelet-derived growth factor (PDGF) promoter, hSYN, the melanin-concentrating hormone (MCH) promoter, CBA, the matrix metalloproteinase promoter (MPP), and the chicken β-actin promoter.

[0058] In addition to the promoter, the vector may also contain one or more other suitable transcription initiation sequences, transcription termination sequences, enhancer sequences, effective RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences stabilizing cytoplasmic mRNA, such as WPRE; sequences enhancing translation efficiency (i.e., Kozak concordant sequences); sequences enhancing protein stability; and sequences enhancing the secretion of the encoded product when desired. An example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include enhancers appropriate for the desired target tissue indication. In one embodiment, the regulatory sequence includes one or more expression enhancers. In one embodiment, the regulatory sequence contains two or more expression enhancers. These enhancers may be the same or different from each other. For example, an enhancer may contain the CMV immediate early enhancer. Such enhancers may be present in two copies located adjacent to each other. Alternatively, the two copies of the enhancer may be separated by one or more sequences. In yet another embodiment, the expression cassette further contains introns, such as chicken β-actin introns. In some embodiments, the intron is a chimeric intron (CI)—a hybrid intron composed of a human β-globin splice donor and an immunoglobulin G (IgG) splice acceptor element. Other suitable introns include introns known in the art, such as those described in WO 2011 / 126808. Examples of suitable polyA sequences include, for example, rabbit globin polyA, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. Optionally, one or more sequences may be selected to stabilize the mRNA. Examples of such sequences are modified WPRE sequences, which may be engineered upstream of the polyA sequence and downstream of the coding sequence (see, for example, MA Zanta-Boussif et al., Gene Therapy (2009) 16:605-619). In some embodiments, the WPRE sequence is not present.

[0059] In some embodiments, the expression cassette refers to a nucleic acid molecule having the nt sequence 213-4439 of SEQ ID NO:1 encoding the amino acid sequence (isotype 1; SEQ ID NO:2) encoding hCDKL5. In some embodiments, the expression cassette refers to a nucleic acid molecule having the nt sequence 213-4562 of SEQ ID NO:5 encoding the amino acid sequence (isotype 2 or 2GS; SEQ ID NO:6) encoding hCDKL5. In some embodiments, the expression cassette refers to a nucleic acid molecule having the nt sequence 213-4388 of SEQ ID NO:7 encoding the amino acid sequence (isotype 3 or 3GS; SEQ ID NO:8) encoding hCDKL5. In some embodiments, the expression cassette refers to a nucleic acid molecule having the nt sequence 213-4511 of SEQ ID NO:9 encoding the amino acid sequence (isotype 4 or 4GS; SEQ ID NO:10) encoding hCDKL5. In some embodiments, the expression cassette includes an engineered nucleic acid sequence selected from SEQ ID NO:37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and encoding an amino acid sequence of hCDKL5 (isotype 1 or SEQ ID NO:2). In some embodiments, the expression cassette refers to a nucleic acid molecule having an amino acid sequence encoding hCDKL5 (isotype 1; SEQ ID NO:4) and including the nt 213-4555 sequence of SEQ ID NO:3 of miRNA183 (SEQ ID NO:11). In some embodiments, these expression cassettes further include one, two, three, four, or more drg-off-target sequences (i.e., miRNAs). See, for example, PCT / US19 / 67872, filed December 20, 2019, and now published as WO 2020 / 132455.

[0060] III.rAAV

[0061] This document provides a recombinant adeno-associated virus (rAAV) for the treatment of CDD. The rAAV comprises: (a) an AAV capsid; and (b) a vector genome packaged within the AAV capsid of (a). Suitably, the selected AAV capsid targets the cells to be treated. In some embodiments, the capsid is derived from clade F. However, in some embodiments, another AAV capsid source may be selected. The vector genome comprises an inverted terminal repeat (ITR) sequence and a nucleic acid sequence encoding a functional human cyclin-dependent kinase-like 5 (hCDKL5), said nucleic acid sequence being under the control of regulatory sequences directing hCDKL5 expression. In some embodiments, CDKL5 may refer to CDKL5 or hCDKL5, CDKL5-2GS or hCDKL5-2GS, CDKL5-3GS or hCDKL5-3GS, and CDKL5-4GS or hCDKL5-4GS. In some embodiments, the hCDKL5 coding sequence is at least about 95% identical to SEQ ID NO:22 (the amino acid sequence encoding CDKL5-1 or hCDKL5-1; SEQ ID NO:2). In some embodiments, the hCDKL5 coding sequence is at least about 95% identical to SEQ ID NO:24 (the amino acid sequence encoding CDKL5-2GS or hCDKL5-2GS; SEQ ID NO:6). In some embodiments, the hCDKL5 coding sequence is at least about 95% identical to SEQ ID NO:25 (the amino acid sequence encoding CDKL5-3GS or hCDKL5-3GS; SEQ ID NO:8). In some embodiments, the hCDKL5 coding sequence is at least about 95% identical to SEQ ID NO:26 (the amino acid sequence encoding CDKL5-4GS or hCDKL5-4GS; SEQ ID NO:10). In some embodiments, the hCDKL5 coding sequence has less than 80% identity with any of the hCDKL5 transcript variants 1 to 3 (NM_001037343.1: having SEQ ID NO:16 and encoding an amino acid sequence having SEQ ID NO:19, NP_001032420.1; NM_001323289.2: having SEQ ID NO:17 and encoding an amino acid sequence having SEQ ID NO:20, NP_001310218.1; NM_003159.2: having SEQ ID NO:18 and encoding an amino acid sequence having SEQ ID NO:21, NP_003150.1).In some embodiments, the hCDKL5 coding sequence is at least about 95% identical to SEQ ID NO:37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47 (encoding CDKL5-1 or hCDKL5-1; the amino acid sequence of SEQ ID NO:2). In some embodiments, the functional hCDKL5 has the amino acid sequence of SEQ ID NO:2 (CDKL5-1 or hCDKL5-1). In some embodiments, the functional hCDKL5 has the amino acid sequence of SEQ ID NO:6 (CDKL5-2GS or hCDKL5-2GS). In some embodiments, the functional hCDKL5 has the amino acid sequence of SEQ ID NO:8 (CDKL5-3GS or hCDKL5-3GS). In some embodiments, the functional hCDKL5 has the amino acid sequence of SEQ ID NO:10 (CDKL5-4GS or hCDKL5-4GS). In some embodiments, the regulatory sequence directs hCDKL5 expression in central nervous system cells. In some embodiments, the regulatory sequence includes the human synaptic protein promoter (hSyn) or the CB7 promoter. In some embodiments, the regulatory element includes one or more of the following: a Kozak sequence, a polyadenylated sequence, an intron, an enhancer, and a TATA signal. In some embodiments, the vector genome further includes at least two tandem repeat sequences of a dorsal root ganglion (drg)-specific miRNA target sequence, wherein the at least two tandem repeat sequences include at least one first miRNA target sequence and at least one second miRNA target sequence, which may be the same or different. In some embodiments, the vector genome is the nt 1 to nt 4634 of SEQ ID NO:1, or the nt 1 to nt 4750 of SEQ ID NO:3, or the nt 1 to nt 4757 of SEQ ID NO:5, or the nt 1 to nt 4583 of SEQ ID NO:7, or the nt 1 to nt 4706 of SEQ ID NO:9, or a nucleic acid sequence that is at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identical to it.

[0062] In some embodiments, the vector genome refers to a nucleic acid molecule comprising the amino acid sequence encoding hCDKL5 (isotype 1; SEQ ID NO:2) of SEQ ID NO:1. In some embodiments, the vector genome refers to a nucleic acid molecule comprising the amino acid sequence encoding hCDKL5 (isotype 2 or 2GS; SEQ ID NO:6) of SEQ ID NO:5. In some embodiments, the vector genome refers to a nucleic acid molecule comprising the amino acid sequence encoding hCDKL5 (isotype 3 or 3GS; SEQ ID NO:8) of SEQ ID NO:7. In some embodiments, the vector genome refers to a nucleic acid molecule comprising the amino acid sequence encoding hCDKL5 (isotype 4 or 4GS; SEQ ID NO:10) of SEQ ID NO:9. In some embodiments, the vector genome refers to a nucleic acid molecule comprising the amino acid sequence encoding hCDKL5 (isotype 1; SEQ ID NO:4) and also comprising miRNA183 (SEQ ID NO:11) of SEQ ID NO:3.

[0063] In some embodiments, in addition to the hCDKL5 coding sequence, another non-AAV coding sequence may be included, such as a peptide, polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. Useful gene products may include miRNAs. miRNAs and other small interfering nucleic acids regulate gene expression through target RNA transcript cleavage / degradation or translational repression of target messenger RNA (mRNA). miRNAs are naturally expressed, typically as the final 19-25 untranslated RNA products. miRNAs exhibit their activity through sequence-specific interactions with the 3' untranslated region (UTR) of the target mRNA. These endogenously expressed miRNAs form hairpin precursors, which are subsequently processed into miRNA duplexes and further processed into "mature" single-stranded miRNA molecules. This mature miRNA guides the multi-protein complex miRISC, which identifies the target site of the target mRNA, such as in the 3'UTR region, based on complementarity with the mature miRNA.

[0064] As used herein, a “miRNA target sequence” is a sequence located on the positive strand of DNA (5' to 3') and at least partially complementary to a miRNA sequence containing a miRNA seed sequence. The miRNA target sequence is exogenous to the untranslated region of the encoded transgenic product and is engineered to be specifically targeted by the miRNA in cells where transgenic expression is to be suppressed. The term “miR183 cluster target sequence” refers to a target sequence that responds to one or more members of the miR183 cluster (or alternatively, the family), including miR-183, miR-96, and miR-182 (as described in Dambal, S. et al., Nucleic Acid Research 43:7173-7188, 2015, which is incorporated herein by reference).

[0065] Typically, the miRNA target sequence is at least 7 to about 28 nucleotides long, at least 8 to about 28 nucleotides long, 7 to 28 nucleotides long, 8 to 18 nucleotides long, 12 to 28 nucleotides long, about 20 to about 26 nucleotides long, about 22 nucleotides long, about 24 nucleotides long, or about 26 nucleotides long, and the miRNA target sequence contains at least one contiguous region (e.g., 7 or 8 nucleotides) complementary to the miRNA seed sequence. In some embodiments, the target sequence includes a sequence that is exactly complementary (100%) or partially complementary to the miRNA seed sequence and has some mismatches. In some embodiments, the target sequence includes at least 7 to 8 nucleotides that are 100% complementary to the miRNA seed sequence. In some embodiments, the target sequence consists of a sequence that is 100% complementary to the miRNA seed sequence. In some embodiments, the target sequence contains multiple copies (e.g., two or three copies) of a sequence that is 100% complementary to the seed sequence. In some embodiments, the 100% complementary region includes at least 30% of the length of the target sequence. In some embodiments, the remaining portion of the target sequence has at least about 80% to about 99% complementarity with the miRNA. In some embodiments, in an expression cassette containing the positive strand of DNA, the miRNA target sequence is the inverse complement of the miRNA.

[0066] As used herein, a “miRNA target sequence” is a sequence located on the positive strand of DNA (5' to 3') and at least partially complementary to a miRNA sequence containing a miRNA seed sequence. The miRNA target sequence is exogenous to the untranslated region of the encoded transgenic product and is engineered to be specifically targeted by the miRNA in cells where transgenic expression is to be suppressed. The term “miR183 cluster target sequence” refers to a target sequence that responds to one or more members of the miR183 cluster (or alternatively, the family), including mir-183, mir-96, and mir-182 (as described in Dambal, S. et al., Nucleic Acid Research 43:7173-7188, 2015, which is incorporated herein by reference). To avoid being bound by theory, messenger RNA (mRNA) used for transgenesis (encoding gene products) exists in cell types that deliver expression cassettes containing miRNAs. This allows the miRNA to specifically bind to the 3'UTR miRNA target sequence, leading to mRNA silencing and cleavage, thereby reducing or eliminating transgene expression only in cells expressing miRNAs.

[0067] Typically, the miRNA target sequence is at least 7 to about 28 nucleotides long, at least 8 to about 28 nucleotides long, 7 to 28 nucleotides long, 8 to 18 nucleotides long, 12 to 28 nucleotides long, about 20 to about 26 nucleotides long, about 22 nucleotides long, about 24 nucleotides long, or about 26 nucleotides long, and the miRNA target sequence contains at least one contiguous region (e.g., 7 or 8 nucleotides) complementary to the miRNA seed sequence. In some embodiments, the target sequence includes a sequence that is exactly complementary (100%) or partially complementary to the miRNA seed sequence and has some mismatches. In some embodiments, the target sequence includes at least 7 to 8 nucleotides that are 100% complementary to the miRNA seed sequence. In some embodiments, the target sequence consists of a sequence that is 100% complementary to the miRNA seed sequence. In some embodiments, the target sequence contains multiple copies (e.g., two or three copies) of a sequence that is 100% complementary to the seed sequence. In some embodiments, the 100% complementary region includes at least 30% of the length of the target sequence. In some embodiments, the remaining portion of the target sequence has at least about 80% to about 99% complementarity with the miRNA. In some embodiments, in an expression cassette containing the positive strand of DNA, the miRNA target sequence is the inverse complement of the miRNA.

[0068] In some embodiments, the miRNA target sequence of at least the first and / or at least the second miRNA target sequence of the expression cassette mRNA or DNA positive strand is selected from: (i) AGTGAATCTACCAGTGCCATA (miR183, SEQ ID NO:11); (ii) AGCAAAAAATGTGCTAGTGCCAAA (miR-96, SEQ ID NO:12); (ii) AGTTGTGAGTTCTACCATTGCCAAA (miR182, SEQ ID NO:13). In other embodiments, AGTGATTCCTGGGAAAACTGGAC (SEQ ID NO:14) is selected.

[0069] In some embodiments, the vector genome or expression cassette contains at least one miRNA target sequence as a miR-183 target sequence. In some embodiments, the vector genome or expression cassette contains a miR-183 target sequence comprising AGTGAATCTACCAGTGCCATA (SEQ ID NO:11), wherein the sequence complementary to the miR-183 seed sequence is GTGCCAT. In some embodiments, the vector genome or expression cassette contains more than one copy (e.g., two or three copies) of a sequence that is 100% complementary to the miR-183 seed sequence. In some embodiments, the miR-183 target sequence is from about 7 nucleotides to about 28 nucleotides in length and contains at least one region that is at least 100% complementary to the miR-183 seed sequence. In some embodiments, the miR-183 target sequence contains a sequence that is partially complementary to SEQ ID NO:11, and therefore, when aligned with SEQ ID NO:11, one or more mismatches exist. In some embodiments, when compared with SEQ ID NO:11, the miR-183 target sequence includes sequences having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, wherein said mismatches may be discontinuous. In some embodiments, the miR-183 target sequence includes a region having 100% complementarity, said region further comprising at least 30% of the length of the miR-183 target sequence. In some embodiments, the region having 100% complementarity includes a sequence having 100% complementarity to the miR-183 seed sequence. In some embodiments, the remainder of the miR-183 target sequence has at least about 80% to about 99% complementarity to miR-183. In some embodiments, the expression cassette or vector genome includes a miR-183 target sequence comprising a truncated SEQ ID NO:11, i.e., a sequence lacking at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at either end or both ends of the 5' or 3' end of SEQ ID NO:1. In some embodiments, the expression cassette or vector genome includes a transgene and a miR-183 target sequence. In still other embodiments, the expression cassette or vector genome includes at least two, three, or four miR-183 target sequences: (i) AGTGAATTCTACCAGTGCCATA (miR183, SEQ ID NO:11); (ii) AGCAAAAATGTGCTAGTGCCAAA (miR-96, SEQ ID NO:12).

[0070] In some embodiments, the vector genome or expression cassette contains at least one miRNA target sequence as a miR-182 target sequence. In some embodiments, the vector genome or expression cassette contains a miR-182 target sequence comprising AGTGTGAGTTCTACCATTGCCAAA (SEQ ID NO:13). In some embodiments, the vector genome or expression cassette contains more than one copy (e.g., two or three copies) of a sequence that is 100% complementary to the miR-182 seed sequence. In some embodiments, the miR-182 target sequence is from about 7 nucleotides to about 28 nucleotides in length and contains at least one region that is at least 100% complementary to the miR-182 seed sequence. In some embodiments, the miR-182 target sequence contains a sequence that is partially complementary to SEQ ID NO:13, and therefore, when compared with SEQ ID NO:13, one or more mismatches exist. In some embodiments, when compared with SEQ ID NO:13, the miR-183 target sequence includes sequences having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, wherein said mismatches may be discontinuous. In some embodiments, the miR-182 target sequence includes a region with 100% complementarity, said region further comprising at least 30% of the length of the miR-182 target sequence. In some embodiments, the region with 100% complementarity includes a sequence that is 100% complementary to the miR-182 seed sequence. In some embodiments, the remainder of the miR-182 target sequence has at least about 80% to about 99% complementarity to miR-182. In some embodiments, the expression cassette or vector genome includes a miR-182 target sequence comprising a truncated SEQ ID NO:13, i.e., a sequence lacking at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at either or both ends of the 5' or 3' end of SEQ ID NO:13. In some embodiments, the expression cassette or vector genome includes a transgene and a miR-182 target sequence. In other embodiments, the expression cassette or vector genome includes at least two, three, or four miR-182 target sequences.

[0071] As used herein, the term "tandem repeat sequence" refers to the presence of two or more consecutive miRNA target sequences. These miRNA target sequences can be consecutive, i.e., directly located one after another, such that the 3' end of one target sequence is directly upstream of the 5' end of the next target sequence, without intermediate sequences, or vice versa. In another embodiment, two or more miRNA target sequences in the miRNA target sequence are separated by short spacer sequences.

[0072] As used herein, a “spacer” is any chosen nucleic acid sequence, for example, a nucleic acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length located between two or more consecutive miRNA target sequences. In some embodiments, the spacer is 1 to 8 nucleotides long, 2 to 7 nucleotides long, 3 to 6 nucleotides long, 4 nucleotides long, 4 to 9 nucleotides long, 3 to 7 nucleotides long, or a greater value. Suitably, the spacer is a non-coding sequence. In some embodiments, the spacer may have four (4) nucleotides. In some embodiments, the spacer is GGAT. In some embodiments, the spacer is six (6) nucleotides. In some embodiments, the spacer is CACGTG or GCATGC.

[0073] In some embodiments, the tandem repeat sequence contains two, three, four, or more miRNA target sequences from the same miRNA target sequence. In some embodiments, the tandem repeat sequence contains at least two, at least three, or at least four different miRNA target sequences. In some embodiments, the tandem repeat sequence may contain two or three of the same miRNA target sequences and a different fourth miRNA target sequence.

[0074] In some embodiments, the expression cassette may contain at least two different sets of tandem repeat sequences. For example, the 3' UTR may contain a tandem repeat sequence immediately downstream of the transgene, a UTR sequence, and two or more tandem repeat sequences closer to the 3' end of the UTR. In another example, the 5' UTR may contain one, two, or more miRNA target sequences. In yet another example, a tandem repeat sequence may be present at the 3' position, and the 5' UTR may contain at least one miRNA target sequence.

[0075] In some embodiments, the expression cassette contains two, three, four, or more tandem repeat sequences that begin within about 0 to 20 nucleotides of the stop codon of the transgene. In other embodiments, the expression cassette contains a miRNA tandem repeat sequence at least 100 to about 4000 nucleotides from the stop codon of the transgene.

[0076] In some embodiments, the spacers between the miRNA target sequences are identical. As used herein, unless otherwise specified, CDKL5 or hCDKL5 refers to isotype 1. Isotypes 2-4 may be designated as: CDKL5-2GS or hCDKL5-2GS, CDKL5-3GS or hCDKL5-3GS, and CDKL5-4GS or hCDKL5-4GS. Expression cassettes and vector genomes having these isotypes can be constructed as described for isotype 1.

[0077] See PCT / US19 / 67872, filed December 20, 2019, now WO 2020 / 132455, which is incorporated herein by reference in its entirety, and U.S. Provisional Patent Application No. 63 / 023,593, filed May 12, 2020; U.S. Provisional Patent Application No. 63 / 038,488, filed June 12, 2020; U.S. Provisional Patent Application No. 63 / 043,562, filed June 24, 2020; and U.S. Provisional Patent Application No. 63 / 079,299, filed September 16, 2020, and U.S. Provisional Patent Application No. 63 / 152,042, filed February 22, 2011, all of which are incorporated herein by reference.

[0078] In some embodiments, the clade F AAV capsid is selected from the AAVhu68 capsid, AAV9 capsid, AAVhu31 capsid, AAVhu32 capsid, or engineered variants of one of these capsids (e.g., AAV-PHP.B). The nucleic acid sequence encoding the AAVhu68 capsid protein is used in examples to generate AAV.hCDKL5 recombinant AAV (rAAV) carrying the vector genome. Further details relating to AAVhu68 or AAV-PHP.B are provided in WO 2018 / 160582 and US 2015 / 0079038, each of which is incorporated herein by reference in its entirety. The clade F vectors described herein are well-suited for delivering vector genomes including the hCDKL5 coding sequence to cells within the central nervous system, which includes the brain, hippocampus, motor cortex, cerebellum, and motor neurons. These vectors can be used to target other cells within the central nervous system (CNS) as well as certain other tissues and cells outside the CNS.

[0079] In some embodiments, the AAV capsid used in the compositions and methods described herein is selected based on target cells. In some embodiments, the AAV capsid transduces CNS cells and / or PNS cells. In some embodiments, the AAV capsid is selected from cy02 capsid, rh43 capsid, AAV8 capsid, rh01 capsid, AAV9 capsid, rh8 capsid, rh10 capsid, bb01 capsid, hu37 capsid, rh02 capsid, rh20 capsid, rh39 capsid, rh64 capsid, AAV6 capsid, AAV1 capsid, hu44 capsid, hu48 capsid, cy05 capsid, hu11 capsid, hu32 capsid, pi2 capsid, or variants thereof. In some embodiments, the AAV capsid is a clade F capsid, such as AAV9 capsid, AAVhu68 capsid, hu31 capsid, hu32 capsid, or variants thereof. See, for example, WO 2005 / 033321, WO 2018 / 160582 and US 2015 / 0079038, published on April 14, 2015, each of which is incorporated herein by reference in its entirety. In some embodiments, the AAV capsid is a non-clade F capsid, such as clades A, B, C, D or E capsids. In some embodiments, the non-clade F capsid is AAV1 or a variant thereof. In some embodiments, the AAV capsid transduces target cells other than neural system cells. In some embodiments, the AAV capsid is a clade A capsid (e.g., AAV1, AAV6, AAVrh91), a clade B capsid (e.g., AAV2), a clade C capsid (e.g., hu53), a clade D capsid (e.g., AAV7) or a clade E capsid (e.g., rh10). In some embodiments, the AAV capsid is a clade A capsid, such as the AAVrh91 capsid (nucleic acid sequences of SEQ ID NO:33 and SEQ ID NO:35). See PCT / US20 / 030266, filed April 29, 2020; WO2020 / 223231, now published, which is incorporated herein by reference; and U.S. Provisional Patent Application No. 63 / 065,616, filed April 29, 2019, which is hereby incorporated by reference. See also U.S. Provisional Application No. 63 / 065,616, filed August 14, 2020, and U.S. Provisional Patent Application No. 63 / 109,734, filed November 4, 2020, which are incorporated herein by reference. Other AAV capsids may also be selected.

[0080] As used herein, the term "clade" in relation to AAV groups refers to a group of phylogenetically related AAVs identified using a neighbor-joining algorithm, based on alignments of the AAV vp1 amino acid sequence, with at least 75% bootstrap values ​​(of at least 1000 copies) and a Poisson correction distance measurement not greater than 0.05. The neighbor-joining algorithm has been described in the literature. See, for example, M. Nei and S. Kumar, *Molecular Evolution and Phylogenetics* (Oxford University Press, New York (2000)). Available computer programs for implementing this algorithm are provided. For example, the MEGA v2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, along with the sequence of the AAV vp1 capsid protein, those skilled in the art can readily determine whether the selected AAV is contained in one of the clades identified herein, or in another clade outside of these clades. See, for example, G Gao et al., *Journal of Virology*, June 2004; 78(10):6381-6388, which identifies clades A, B, C, D, E, and F and provides nucleic acid sequences of novel AAVs, GenBank accessions AY530553 to AY530629. See also WO2005 / 033321.

[0081] rAAV consists of an AAV capsid and a vector genome. The AAV capsid is an assembly of heterogeneous populations of vp1, vp2, and vp3 proteins. As used herein, when referring to vp capsid proteins, the term "heterogeneous" or any grammatical variation thereof refers to a population of dissimilar elements, such as vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences.

[0082] As used herein, when referring to VP capsid proteins, the term "heterogeneous" or any grammatical variation thereof refers to a group of dissimilar elements, such as VP1, VP2, or VP3 monomers (proteins) having different modified amino acid sequences. The term "heterogeneous population," used in conjunction with VP1, VP2, and VP3 proteins (which may alternatively be referred to as isotypes), refers to differences in the amino acid sequences of VP1, VP2, and VP3 proteins within the capsid. The AAV capsid contains subpopulations of VP1, VP2, and VP3 proteins with modifications derived from predicted amino acid residues. These subpopulations contain at least some deamidated asparagine (N or Asn) residues. For example, some subpopulations include at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further include other deamidated amino acids, wherein deamidation causes amino acid changes and other optional modifications.

[0083] In some embodiments, an AAV capsid is provided having a heterogeneous population of AAV capsid isotypes (i.e., VP1, VP2, VP3) containing multiple highly deamidated “NG” positions. In some embodiments, the highly deamidated positions are located at the positions identified in the predicted full-length VP1 amino acid sequence mentioned below. In other embodiments, the capsid gene is modified such that the referenced “NG” is ablated, and a mutant “NG” is engineered to another position.

[0084] As used herein, the terms “target cell” and “target tissue” can refer to any cell or tissue intended to be transduced by the subject’s AAV vector. These terms can refer to any one or more of the following: muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (eye cells), or heart.

[0085] As used herein, the term "vector genome" refers to a nucleic acid molecule packaged in a viral capsid (e.g., an AAV capsid) and capable of being delivered to host or patient cells. In some embodiments, the vector genome is an expression cassette having inverted terminal repeat (ITR) sequences necessary for packaging the vector genome into the AAV capsid at its 5' and 3' ends, and containing therebetween a CDLK5 gene operatively linked to a sequence as described herein to direct its expression. In some embodiments, the vector genome may include at least an AAV 5' ITR from 5' to 3', a coding sequence, and an AAV 3' ITR. In some embodiments, an ITR from AAV2 (an AAV from a different capsid source) or a non-full-length ITR may be selected. In some embodiments, the ITR is from the same AAV source that provides rep functionality during the production or trans-complementation of AAV. Further, other ITRs may be used. The vector genome is sometimes referred to herein as a "small gene".

[0086] As used herein, the term "host cell" can refer to the packaging cell line for rAAV produced by a plasmid. Alternatively, the term "host cell" can refer to the target cell for which transgene expression is desired.

[0087] As noted above, rAAV is provided with an AAV capsid and a vector genome targeting desired cells, said vector genome comprising at least the AAV ITR, hCDKL5 coding sequences required for packaging the vector genome into the capsid, and regulatory sequences guiding its expression. In some embodiments, the vector genome is a single-stranded AAV vector genome. In some embodiments, an rAAV vector containing a self-complementary (sc) AAV vector genome may be used in this invention.

[0088] The AAV sequence of the vector typically includes cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (see, for example, B.J. Carter, Handbook of Parvoviruses, edited by P. Tijsser, CRC Press, pp. 155–168 (1990)). The ITR sequence is approximately 145 base pairs (bp) long. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, although minor modifications to these sequences are permitted. The ability to modify these ITR sequences is within the scope of the art. (See, for example, texts such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., Journal of Virology, 70:520–532 (1996)). An example of such a molecule used in this invention is a transgenic "cis-acting" plasmid, wherein the selected transgenic sequence and associated regulatory element are flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITR originates from an AAV different from the AAV supplying the capsid. In one embodiment, the ITR sequence originates from AAV2. A shortened version of the 5' ITR, referred to as ΔITR, has been described, in which the D sequence and terminal resolution site (trs) are missing. In other embodiments, full-length AAV 5' and 3' ITRs are used. In some embodiments, the vector genome contains a shortened 130-base-pair AAV2 ITR, in which the outer A element is missing. During vector DNA amplification using the inner A element as a template, the shortened ITR reverts to a wild-type length of 145 base pairs. However, ITRs from other AAV sources can be selected. In cases where the ITR originates from AAV2 and the AAV capsid originates from another AAV source, the resulting vector can be referred to as pseudotyped. However, other configurations of these elements can be suitable.

[0089] In some embodiments, a vector genome comprising a 5'AAV ITR-promoter-optional enhancer-optional intron-hCDKL5 coding sequence-polyA-3'ITR is constructed. In some embodiments, a vector genome comprising a 5'AAV ITR-promoter-optional enhancer-optional intron-hCDKL5 coding sequence-optionally repeating miR off-target sequence-polyA-3'ITR is constructed. In some embodiments, a vector genome comprising a 5'AAV ITR-promoter-optional intron-hCDKL5 coding sequence-optional enhancer-polyA-3'ITR is constructed. In some embodiments, a vector genome comprising a 5'AAVITR-promoter-optional enhancer-optional intron-hCDKL5 coding sequence-optional enhancer-optionally repeating miR off-target sequence-polyA-3'ITR is constructed. In some embodiments, the ITR is derived from AAV2. In some embodiments, more than one promoter is present. In some embodiments, the enhancer is present in the vector genome. In some embodiments, more than one enhancer is present. In some embodiments, introns are present in the vector genome. In some embodiments, both enhancers and introns are present. In some embodiments, polyA is SV40polyA (i.e., a polyadenylation (PolyA) signal derived from the late gene of simian virus 40 (SV40). In some embodiments, polyA is rabbit β-globin (RBG) polyA. In some embodiments, the vector genome includes at least: 5'AAV ITR-hSyn promoter-hCDKL5 coding sequence-polyA-3'ITR. In some embodiments, the vector genome includes at least 5'AAV ITR-CB7 promoter-hCDKL5 coding sequence-RBGpolyA-3'ITR. In some embodiments, the drg off-target sequence is one, two, three, four or more miR183 sequences as described herein, and is contained in an expression cassette. In some embodiments, the hCDKL5 coding sequence targets CDKL5. In some embodiments, the hCDKL5 coding sequence targets CDKL5-2GS. In some embodiments, the hCDKL5 coding sequence targets CDKL5-3GS. In some embodiments, the hCDKL5 coding sequence targets CDKL5-4GS. Optionally, one or more of these vector genomes contain WPRE elements.

[0090] As used herein, a vector genome or an rAAV including a vector genome is presented herein as AAV-promoter (optional)-Kozak (optional)-intron (optional)-CDKL5 coding sequence (e.g., hCDKL5, hCDKL5co, CDKL5, CDKL5co)-miRNA (optional)-polyA (optional)-filler fragment (optional). In some embodiments, rAAV is presented herein as AAV capsid-promoter (optional)-Kozak (optional)-intron (optional)-CDKL5 coding sequence-miRNA (optional)-polyA (optional)-filler fragment (optional). Optionally, one or more of these vector genomes contain WPRE elements.

[0091] In some embodiments, the vector genome includes at least a 5'AAV ITR-ubiquitin C promoter-hCDKL5 coding sequence-one, two, three, four or more miR183 sequences-RBG polyA-3'ITR. In some embodiments, the vector genome includes at least a 5'AAV ITR-chicken β-actin promoter-hCDKL5 coding sequence-one, two, three, four or more miR183 sequences-RBG polyA-3'ITR. Optionally, one or more of these vector genomes contain WPRE elements.

[0092] Additionally, this document provides an rAAV generation system that can be used to generate rAAV as described herein. The generation system includes a cell culture comprising (a) a nucleic acid sequence encoding an AAV capsid protein; (b) a vector genome; and (c) sufficient AAV rep functionality and auxiliary functions to allow the vector genome to be packaged into an AAV capsid. In some embodiments, the vector genome is SEQ ID NO: 1, 3, 5, 7, 9, 29, or 31. In some embodiments, the cell culture is a human embryonic kidney 293 cell culture. In some embodiments, the AAV rep is derived from a different AAV. In some embodiments, the AAV rep is derived from AAV2. In some embodiments, the AAV rep coding sequence and the cap gene are on the same nucleic acid molecule, wherein a spacer is optionally present between the rep sequence and the cap gene. In some embodiments, the spacer is atgacttaaaccaggt (SEQ ID NO: 15).

[0093] For the purpose of generating AAV viral vectors (e.g., recombinant (r)AAV), the vector genome can be carried on any suitable vector (e.g., plasmid) delivered to the packaging host cell. Plasmids that can be used in this invention can be engineered to be suitable for in vitro replication and packaging in prokaryotic cells, insect cells, mammalian cells, and other cells. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by those skilled in the art.

[0094] Methods for generating and isolating AAVs suitable for use as vectors are known in the art. See, for example, Grieger and Samulski, 2005, Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al., 2008, Recent developments in adeno-associated virus vector technology, J. Gene Med. 10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety. As used herein, a gene therapy vector refers to rAAV suitable for treating patients as described herein. For packaging genes into viral particles, the ITR is the cis-only AAV component required in the same construct as the nucleic acid molecule containing the gene. The cap and rep genes can be supplied in trans form.

[0095] In one embodiment, the selected genetic element can be delivered to AAV packaging cells by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated granules, viral infection, and protoplast fusion. Stable AAV packaging cells can also be prepared. Methods for preparing such constructs are known to those skilled in nucleic acid manipulation and involve genetic engineering, recombinant engineering, and synthetic techniques. See, for example, *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012).

[0096] The term "AAV intermediate" or "AAV vector intermediate" refers to an assembled rAAV capsid lacking the desired genomic sequence packaged within it. These can also be referred to as "empty" capsids. Such capsids may not contain a detectable genomic sequence of the expression cassette, or may contain only a partially packaged genomic sequence insufficient to achieve expression of the gene product. These empty capsids are non-functional for transferring the gene of interest into the host cell.

[0097] Recombinant adeno-associated virus (AAV) as described herein can be generated using known techniques. See, for example, WO2003 / 042397, WO 2005 / 033321, WO 2006 / 110689, and US 7588772 B2. Such methods involve culturing host cells containing a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette consisting at least of an AAV inverted terminal repeat (ITR) sequence and a transgene; and sufficient auxiliary functions to allow the expression cassette to be packaged into the AAV capsid protein. Methods for generating the capsid, its coding sequence, and methods for generating rAAV viral vectors have been described. See, for example, Gao et al., Proceedings of the National Academy of Sciences, 100(10), 6081-6086 (2003) and US2013 / 0045186A1.

[0098] In one embodiment, a generative cell culture for generating recombinant AAVhu68 or AAVrh91 is provided. Such a cell culture contains nucleic acids expressing the AAVhu68 capsid protein in host cells; nucleic acid molecules suitable for packaging into the AAVhu68 capsid, such as a vector genome containing an AAV ITR and a non-AAV nucleic acid sequence encoding a gene operatively linked to a regulatory sequence that directs the expression of the gene in the host cell; and AAV rep functionality and adenovirus helper functionality sufficient to allow the vector genome to be packaged into the recombinant AAVhu68 or AAVrh91 capsid. In one embodiment, the cell culture comprises mammalian cells (e.g., human embryonic kidney 293 cells and other cells) or insect cells (e.g., fall armyworm (Spodoptera frugiperda) (Sf9) cells). In some embodiments, a baculovirus provides the helper functionality necessary for packaging the vector genome into the recombinant AAVhu68 or AAVrh91 capsid.

[0099] Optionally, the rep function is provided by an AAV other than hu68 or AAV-PHP.B. In some embodiments, at least a portion of the rep function originates from AAVhu68 or AAVrh91. In another embodiment, the rep protein is a heterologous rep protein other than AAVhu68rep, such as, but not limited to, AAV1 rep, AAV2 rep, AAV3 rep, AAV4 rep, AAV5 rep, AAV6 rep, AAV7 rep, AAV8 rep; or rep 78, rep 68, rep 52, rep 40, rep 68 / 78, and rep 40 / 52; or fragments thereof; or other sources. Any of these AAVhu68 or mutant AAV capsid sequences can be under the control of exogenous regulatory sequences that direct their expression in host cells.

[0100] In one embodiment, cells are manufactured in a suitable cell culture (e.g., HEK 293 or Sf9) or suspension. Methods for manufacturing the gene therapy vectors described herein include methods well-known in the art, such as generating plasmid DNA for producing the gene therapy vector, generating the vector, and purifying the vector. In some embodiments, the gene therapy vector is an AAV vector, and the generated plasmid is an AAV cis plasmid encoding the AAV vector genome and the gene of interest, an AAV trans plasmid containing the AAV rep and cap genes, and an adenovirus helper plasmid. The vector production process may include method steps such as initiating cell culture, performing cell passage, seeding cells, transfecting cells with plasmid DNA, exchanging the transfected medium for serum-free medium, and collecting the vector-containing cells and medium. The collected vector-containing cells and medium are referred to herein as crude cell collection. In yet another system, the gene therapy vector is introduced into insect cells by infection with a baculovirus-based vector. For reviews of these production systems, see, for example, Zhang et al., 2009, Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929. The contents of each of these references are incorporated herein by reference in full. Methods for preparing and using these and other AAV generation systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entirety: U.S. Patent Nos. 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065.

[0101] Subsequently, the crude cell collection can be prepared using the methodological steps of this subject, such as concentrating the carrier collection, percolating the carrier collection, microfluidizing the carrier collection, digesting the carrier collection with nucleases, filtering the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange and / or formulation and filtration by tangential flow filtration to prepare a large number of carriers.

[0102] Two-step affinity chromatography purification was performed at high salt concentrations, followed by anion exchange resin chromatography to purify the carrier drug product and remove empty capsids. These methods are described in more detail in WO 2017 / 160360, filed December 9, 2016, and its priority document; U.S. Patent Application No. 62 / 322,071, filed April 13, 2016; and U.S. Patent Application No. 62 / 226,357, filed December 11, 2015, entitled “Scalable Purification Method for AAV9,” which are incorporated herein by reference. In some embodiments, the purification of the carrier drug product (e.g., AAVrh91) comprises the purification of the carrier drug product described in WO2017 / 100674, filed December 9, 2016, and its priority document; U.S. Provisional Patent Application No. 62 / 266,351, filed December 9, 2015; and U.S. Patent Application No. 62 / 322,083, filed April 13, 2016, entitled “Scalable Purification Method for AAV1,” which are incorporated herein by reference.

[0103] To calculate the content of empty and full particles, the VP3 band volume of the selected sample (e.g., in the examples of this paper, a formulation purified by iodixanol gradient purification, where the number of genome copies (GC) = the number of particles) was plotted against the loaded GC particles. The resulting linear equation (y = mx + c) was used to calculate the number of particles in the band volume of the test peak. The number of particles per 20 μL loaded (pt) was then multiplied by 50 to obtain particles (pt) / mL. Pt / mL was divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL - GC / mL gave the empty pt / mL. Empty pt / mL was divided by pt / mL and multiplied by 100 to obtain the percentage of empty particles.

[0104] Typically, methods for determining empty capsids and AAV vector particles containing packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molecular Therapy (2003) 7:122-128. To test for denatured capsids, the method comprises subjecting a treated AAV stock solution to SDS-polyacrylamide gel electrophoresis (consisting of any gel capable of separating the three capsid proteins, such as a gradient gel containing 3-8% triacetate in buffer), then running the gel until sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane (preferably nylon). An anti-AAV capsid antibody is then used as a primary antibody to bind to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., Journal of Virology (2000) 74:9281-9293). Then, a secondary antibody is used, which binds to the primary antibody and contains a device for detecting the binding to the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound thereto, and most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting the binding is used to semi-quantitatively determine the binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioisotope emission, electromagnetic radiation, or colorimetric changes, and most preferably a chemiluminescent detection kit. For example, for SDS-PAGE, the sample can be extracted from the column fraction and heated in an SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and the capsid protein can be resolved on a pre-prepared gradient polyacrylamide gel (e.g., Novex). Silver staining can be performed using SilverXpress (Invitrogen, CA) or other suitable staining methods (i.e., SYPRO ruby ​​or Coomassie staining) according to the manufacturer's instructions. In one embodiment, the concentration of the AAV vector genome (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample was diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, primers and TaqMan sequences specific to the DNA sequence between the primers were used. TMThe fluorescent probes were further diluted and amplified in the samples. The number of cycles (threshold cycles, Ct) required for each sample to reach a defined fluorescence level was measured on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector was used to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample was used to determine the vector genomic titer by normalizing it relative to the Ct value of the plasmid standard curve. Endpoint assays based on digital PCR can also be used.

[0105] On the one hand, an optimized q-PCR method was used, which utilizes a broad-spectrum serine protease, such as proteinase K (commercially available from Qiagen). More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, followed by heat inactivation. Suitablely, the sample is diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer can be concentrated 2-fold or more. Typically, the proteinase K treatment is about 0.2 mg / mL, but it can vary between 0.1 g / mL and about 1 mg / mL. The treatment step is typically performed at about 55°C for about 15 minutes, but it can be performed at lower temperatures (e.g., about 37°C to about 50°C) for a longer period of time (e.g., about 20 minutes to about 30 minutes), or at higher temperatures (e.g., up to about 60°C) for a shorter period of time (e.g., about 5 to 10 minutes). Similarly, thermal inactivation typically occurs at approximately 95°C for about 15 minutes, but the temperature can be lowered (e.g., from approximately 70°C to approximately 90°C) and the time extended (e.g., from approximately 20 minutes to approximately 30 minutes). The sample is then diluted (e.g., 1000-fold) and analyzed using TaqMan as described in the standard assay.

[0106] Alternatively or alternatively, droplet digital PCR (ddPCR) can be used. For example, methods for determining the genomic titer of single-stranded and self-complementary AAV vectors by ddPCR have been described. See, for example, M. Lock et al., Human Gene Therapy Methods. April 2014; 25(2):115-25. doi:10.1089 / hgtb.2013.131. Electronic publication February 14, 2014.

[0107] In summary, the method for isolating rAAVhu68 (or AAVrh91) particles with packaged genomic sequences from genomically defective AAVhu68 (or AAVrh91) intermediates involves subjecting a suspension comprising recombinant AAVhu68 (or rh91) viral particles and AAVhu68 (or AAVrh91) capsid intermediates to high-performance liquid chromatography (HPLC), wherein the AAVhu68 (or AAVrh91) viral particles and AAVhu68 intermediates are bound to a strong anion exchange resin equilibrated at approximately 10.2 (or approximately 9.8 for AAVrh91) and subjected to a salt gradient, while monitoring the UV absorbance of the eluent at approximately 260 nanometers (nm) and approximately 280 nm. Although not optimal for rAAVhu68 and AAVrh91, the pH range can be approximately 10 to 10.4. In this method, when the A260 / A280 ratio reaches an inflection point, the intact AAV capsid is collected from the eluted fraction. In one example, for the affinity chromatography step, the percolated product can be applied to an affinity resin (Life Technologies) that effectively captures the AAVhu68 or AAVrh91 serotypes. Under these ionic conditions, a significant percentage of residual cellular DNA and protein flows through the column, while AAV particles are effectively captured.

[0108] rAAV.hCDKL5 is suspended in a suitable physiologically compatible composition (e.g., buffered saline). This composition can be cryopreserved, then thawed, and optionally diluted with a suitable diluent. Alternatively, the carrier can be prepared as a composition suitable for delivery to patients without the need for freezing and thawing steps.

[0109] As used herein, the term “NAb titer” is a measure of how many neutralizing antibodies (e.g., anti-AAV Nab) are produced that neutralize the physiological effects of their targeted epitopes (e.g., AAV). Anti-AAV NAb titer can be measured as described, for example, in Calcedo, R et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009, 199(3): 381-390, which is incorporated herein by reference.

[0110] The abbreviation "sc" stands for self-complementary. "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. Upon infection, instead of waiting for cell-mediated synthesis of the second strand, two complementary half-scAAVs associate to form a single double-stranded DNA (dsDNA) that is readily replicated and transcribed. See, for example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis," *Gene Therapy*, (August 2001), Vol. 8, No. 16, pp. 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patents 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0111] "Replication-deficient virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing the gene of interest is packaged within a viral capsid or envelope, wherein any viral genome sequence also packaged within the viral capsid or envelope is replication-deficient; that is, the synthetic or artificial viral particle cannot generate progeny viruses but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless"—containing only the gene of interest, flanked by signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for use in gene therapy because replication and infection via progeny viral particles do not occur unless the viral enzymes required for replication are present.

[0112] In many cases, rAAV particles are referred to as DNase-resistant. However, in addition to this nuclease (DNase), other nucleases and exonucleases can also be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases can be selected to degrade single-stranded DNA and / or double-stranded DNA, as well as RNA. These steps can contain a single nuclease or a mixture of nucleases targeting different targets, and can be either endonucleases or exonucleases.

[0113] The term "nuclease resistance" indicates that the AAV capsid has been fully assembled around the expression cassette, which is designed to deliver genes to host cells and protect the packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove contaminating nucleic acids that may be present during the production process.

[0114] IV. Other carriers

[0115] This document provides vectors comprising expression cassettes as described herein. In some embodiments, the expression cassette comprises a nucleic acid sequence encoding a functional human cyclin-dependent kinase-like 5 (hCDKL5), said nucleic acid sequence being under the control of regulatory sequences directing hCDKL5 expression. In some embodiments, the hCDKL5 coding sequence encodes the hCDKL5 protein, said hCDKL5 protein comprising...

[0116] the amino acid sequence (SEQ ID NO: 2) of [MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRSAKRKPYHVESSTLSNRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETAL],The hCDKL5 coding sequence is the nucleic acid sequence of SEQ ID NO:22 or has a sequence that is at least about 95% identical to SEQ ID NO:22 and encodes a protein of SEQ ID NO:2. In some embodiments, the hCDKL5 coding sequence encodes the hCDKL5-2GS protein, which comprises,

[0117]

[0118] [MKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTLRELKMLRTLKQENIVELKEAFRRRGKLYLVFEYVEKNMLELLEEMPNGVPPEKVKSYIYQLIKAIHWCHKNDIVHRDIKPENLLISHNDVLKLCDFGFARNLSEGNNANYTEYVATRWYRSPELLLGAPYGKSVDMWSVGCILGELSDGQPLFPGESEIDQLFTIQKVLGPLPSEQMKLFYSNPRFHGLRFPAVNHPQSLERRYLGILNSVLLDLMKNLLKLDPADRYLTEQCLNHPTFQTQRLLDRSPSRNQAGKSTALQSHHRSNSKDIQNLSVGLPRADEGLPANESFLNGNLAGASLSPLHTKTYQASSQPGSTSKDLTNNNIPHLLSPKEAKSKTEFDFNIDPKPSEGPGTKYLKSNSRSQQNRHSFMESSQSKAGTLQPNEKQSRHSYIDTIPQSSRSPSYRTKAKSHGALSDSKSVSNLSEARAQIAEPSTSRYFPSSCLDLNSPTSPTPTRHSDTRTLLSPSGRNNRNEGTLDSRRTTTRHSKTMEELKLPEHMDSSHSHSLSAPHESFSYGLGYTSPFSSQQRPHRHSMYVTRDKVRAKGLDGSLSIGQGMAARANSLQLLSPQPGEQLPPEMTVARSSVKETSREGTSSFHTRQKSEGGVYHDPHSDDGTAPKENRHLYNDPVPRRVGSFYRVPSPRPDNSFHENNVSTRVSSLPSESSSGTNHSKRQPAFDPWKSPENISHSEQLKEKEKQGFFRSMKKKKKKSQTVPNSDSPDLLTLQKSIHSASTPSSRPKEWRPEKISDLQTQSQPLKSLRKLLHLSSASNHPASSDPRFQPLTAQQTKNSFSEIRIHPLSQASGGSSNIRQEPAPKGRPALQLPGQMDPGWHVSSVTRSATEGPSYSEQLGAKSGPNGHPYNRTNRSRMPNLNDLKETAL] the amino acid sequence of (SEQ ID NO:8),The hCDKL5 coding sequence comprises the nucleic acid sequence of SEQ ID NO:25 or a sequence that is at least about 95% identical to SEQ ID NO:25 and encodes the protein of SEQ ID NO:8. In some embodiments, the hCDKL5 coding sequence encodes the hCDKL5-4GS protein, said hCDKL5-4GS comprising,

[0119] the amino acid sequence (SEQ IDNO:10), wherein the hCDKL5 coding sequence comprises the nucleic acid sequence of SEQ ID NO:26 or a sequence that is at least about 95% identical to SEQ ID NO:26 and encodes the protein of SEQ ID NO:10.

[0120] In some embodiments, the vector is a viral vector selected from recombinant parvovirus, recombinant lentivirus, recombinant retrovirus, or recombinant adenovirus; or a non-viral vector selected from naked DNA, naked RNA, inorganic particles, lipid particles, polymer-based vectors, or chitosan-based formulations. The selected vector can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion, high-speed DNA-coated granules, viral infection, and protoplast fusion. Methods for preparing such constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombination engineering, and synthetic techniques. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

[0121] "Replication-deficient virus" or "viral vector" refers to a synthetic or artificial viral particle containing an expression cassette of the gene of interest packaged within a viral capsid or envelope, wherein any viral genome sequence also packaged within the viral capsid or envelope is replication-deficient; that is, the synthetic or artificial viral particle cannot generate progeny viruses but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless"—containing only the transgene of interest, which is flanked by signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for use in gene therapy because replication and infection via progeny viral particles do not occur unless the viral enzymes required for replication are present. Such replication-deficient viruses can be adeno-associated virus (AAV), adenovirus, lentivirus (integrating or non-integrating), or other suitable viral sources.

[0122] V. Composition

[0123] This document provides a composition comprising rAAV or carrier as described herein and an aqueous suspension medium. In some embodiments, the suspension is formulated for intravenous delivery, intrathecal administration, or intraventricular administration.

[0124] This document provides compositions containing at least one rAAV stock solution and optional carrier, excipients, and / or preservatives. As used herein, "stock solution" of rAAV refers to a population of rAAVs. Although the capsid proteins of rAAVs are heterogeneous due to deamidation, the rAAVs in the stock solution are expected to share the same vector genome. The stock solution may contain rAAVs with a capsid having, for example, a heterogeneous deamidation pattern specific to the selected AAV capsid protein and the selected production system. The stock solution may be produced from a single production system or pooled from multiple runs of a production system. Various production systems may be selected, including but not limited to those described herein.

[0125] As used herein, "carrier" includes any and all solvents, dispersion media, mordants, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption-delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such culture media and agents for pharmaceutically active substances is well known in the art. Complementary active ingredients may also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic reactions or similar adverse reactions when administered to a host. Delivery media (such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc.) can be used to introduce the compositions of the present invention into suitable host cells. Specifically, the vector genome delivered by the rAAV vector can be formulated for delivery or encapsulation in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc.

[0126] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, said composition being, for example, an aqueous liquid suspension buffered to physiologically compatible pH and salt concentrations. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.

[0127] Suitable surfactants or combinations of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminated at a primary hydroxyl group is selected, such as... F68 [BASF], also known as Poloxamer 188, has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers, i.e., nonionic triblock copolymers, can be selected, consisting of a central hydrophobic chain of polyoxypropylene (poly(ethylene oxide)) attached to two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (polyethylene glycol-15-hydroxystearate), LABRASOL (polyoxyethyl octanoate), polyoxy 10 oleate ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains poloxamer. These copolymers are typically named with the letter "P" (for poloxamers), followed by three numbers: the first two digits × 100 give the approximate molecular weight of the polyoxypropylene core, and the last digit × 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 was selected. In one embodiment, the surfactant may be present in an amount of up to about 0.0005% to about 0.001% (w / w%) of the suspension. In another embodiment, the surfactant may be present in an amount of up to about 0.0005% to about 0.001% (w / w%) of the suspension. In yet another embodiment, the surfactant may be present in an amount of up to about 0.0005% to about 0.001% of the suspension, where n% represents n grams per 100 mL of suspension.

[0128] In another embodiment, the composition comprises a carrier, a diluent, an excipient, and / or an adjuvant. Given the indications targeted by the transferred virus, those skilled in the art can readily select a suitable carrier. For example, a suitable carrier comprises saline, which can be formulated with a variety of buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers comprise sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier should contain components that prevent rAAV from adhering to the infusion tubing but do not interfere with rAAV's in vivo binding activity. Suitable surfactants or combinations of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminated at a primary hydroxyl group is selected, such as poloxamer 188 (also trade name). F68 [BASF] F68 F68 P188 (known) has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers, i.e., nonionic triblock copolymers, can be selected, consisting of a central hydrophobic chain of polyoxypropylene (poly(ethylene oxide)) attached to two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (polyethylene glycol-15-hydroxystearate), LABRASOL (polyoxyethyl octanoate), polyoxyethylene oleate, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains poloxamer. These copolymers are typically named with the letter "P" (for poloxamers), followed by three numbers: the first two digits × 100 give the approximate molecular weight of the polyoxypropylene core, and the last digit × 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 was selected. Surfactants may be present in a suspension at a maximum of about 0.0005% to about 0.001%.

[0129] In some embodiments, compositions containing rAAV.hCDKL5 are delivered at a pH in the range of 6.8 to 8, or 7.2 to 7.8, or 7.5 to 8. For intrathecal delivery, a pH higher than 7.5 may be desired, for example, 7.5 to 8, or 7.8.

[0130] In some embodiments, the formulation may contain an aqueous buffer solution that does not contain sodium bicarbonate. Such formulations may contain an aqueous buffer solution comprising one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and mixtures thereof, such as Harvard's buffer. The aqueous solution may further contain… P188, Poloxamer, is available from BASF and was previously sold under the trade name P188. F68 is for sale. The pH of the aqueous solution can be 7.2.

[0131] In another embodiment, the formulation may contain an aqueous buffer solution comprising 1 mM sodium phosphate (Na3PO4), 150 mM sodium chloride (NaCl), 3 mM potassium chloride (KCl), 1.4 mM calcium chloride (CaCl2), 0.8 mM magnesium chloride (MgCl2), and 0.001% poloxamer (e.g., at pH 7.2) at pH 7.2. 188. See, for example, harvardapparatus.com / harvard-apparatus-perfusion-fluid.html. In some embodiments, Harvard buffer is preferred because better pH stability has been observed when using Harvard buffer.

[0132] In some embodiments, the formulation buffer is an artificial CSF with Pluronic F68. In other embodiments, the formulation may contain one or more osmosis enhancers. Examples of suitable osmosis enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium decanoate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0133] Optionally, the compositions of the present invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and one or more carriers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0134] Compositions according to the invention may include pharmaceutically acceptable carriers, as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAVs suspended in a pharmaceutically suitable carrier and / or mixed with a suitable excipient designed for delivery to a subject by injection, osmotic pump, intrathecal catheter, or by another device or route. In one example, the composition is formulated for intrathecal delivery. In one example, the composition is formulated for intravenous (iv) delivery.

[0135] VI. Uses

[0136] This article provides a method for treating CDD, which includes administering an effective amount of rAAV or a vector as described herein to a subject in need.

[0137] In some embodiments, the “effective amount” herein refers to the amount that achieves improvement in CDD symptoms and / or delays CDD progression.

[0138] Administering the vector in sufficient quantities to transfect cells and provide adequate levels of gene transfer and expression to deliver therapeutic benefit without causing excessive side effects or having medically acceptable physiological effects can be determined by a person skilled in the medical field. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., brain, CSF, liver (optionally via hepatic artery), lung, heart, eye, kidney), oral, inhalation, intranasal, intrathecal, intratracheal, intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, intraparenchymal, intravenous, intrathecal, intracranial, intrathecal, ICM, lumbar puncture, and other parenteral routes. Routes of administration may be combined if necessary.

[0139] The dosage of a viral vector (e.g., rAAV) will depend primarily on factors such as the condition being treated, the patient's age, weight, and health status, and therefore may vary between patients. For example, the therapeutically effective human dose of a viral vector typically ranges from about 25 to about 1000 μL to about 100 mL of solution containing a concentration of about 1 × 10⁻⁶. 9 One to 1×10 16 One vector genome copy. In some embodiments, a suspension volume of about 1 mL to about 15 mL, or about 2.5 mL to about 10 mL, or about 5 mL is delivered. In some embodiments, a suspension volume of about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, or about 15 mL is delivered. In some embodiments, a total of about 8.9 × 10⁻⁶ is administered using this volume. 12 Up to 2.7×10 14 GC dosage. In some embodiments, approximately 1.1 × 10⁻⁶ GC is administered in this volume. 10 GC / g brain mass up to approximately 3.3 × 10⁻⁶ 11 Dosage per GC / g brain mass. In some embodiments, the following dose is administered at this volume: approximately 3.0 × 10⁻⁶. 9 Approximately 4.0 × 10 9 Approximately 5.0 × 10 9 Approximately 6.0 × 10 9 Approximately 7.0 × 10 9 Approximately 8.0 × 10 9 Approximately 9.0 × 10 9 Approximately 1.0 × 10 10 Approximately 1.1 × 10 10 Approximately 1.5 × 10 10 Approximately 2.0 × 10 10 Approximately 2.5 × 10 10 Approximately 3.0 × 10 10 Approximately 3.3 × 10 10 Approximately 3.5 × 10 10 Approximately 4.0 × 10 10 Approximately 4.5 × 10 10 Approximately 5.0 × 10 10 Approximately 5.5 × 10 10 Approximately 6.0 × 10 10 Approximately 6.5 × 10 10 Approximately 7.0 × 10 10 Approximately 7.5 × 10 10 Approximately 8.0 × 10 10 Approximately 8.5 × 10 10 Approximately 9.0 × 1010 Approximately 9.5 × 10 10 Approximately 1.0 × 10 11 Approximately 1.1 × 10 11 Approximately 1.5 × 10 11 Approximately 2.0 × 10 11 Approximately 2.5 × 10 11 Approximately 3.0 × 10 11 Approximately 3.3 × 10 11 Approximately 3.5 × 10 11 Approximately 4.0 × 10 11 Approximately 4.5 × 10 11 Approximately 5.0 × 10 11 Approximately 5.5 × 10 11 Approximately 6.0 × 10 11 Approximately 6.5 × 10 11 Approximately 7.0 × 10 11 Approximately 7.5 × 10 11 Approximately 8.0 × 10 11 Approximately 8.5 × 10 11 Approximately 9.0 × 10 11 GC per brain mass.

[0140] Dosage can be adjusted to balance therapeutic benefits with any side effects, and such dosages can vary depending on the therapeutic application employing the recombinant vector. The expression level of the transgenic product can be monitored to determine the dosage frequency of the resulting viral vector, preferably an AAV vector containing a mini-gene. Optionally, dosage regimens similar to those described for therapeutic purposes can be used for immunization with the compositions of the present invention.

[0141] The replication-defective virus composition can be prepared in dosage units to achieve a content of approximately 1.0 × 10⁻⁶ replication-defective virus. 9 GC to approximately 1.0 × 10 16 The range of GC (for treating subjects) includes all integers or fractions within the range, and for human patients, it is preferably 1.0 × 10⁻⁶. 12 GC up to 1.0×10 14 GC. In one embodiment, the composition is formulated to contain at least 1 × 10⁻⁶ GC per dose. 9 2×10 9 3×10 9 4×10 9 5×10 9 6×10 9 7×10 9 8×10 9 Or 9×10 9GC includes all integers or fractions within the range. In another embodiment, the composition is formulated to contain at least 1 × 10⁻⁶ per dose. 10 2×10 10 3×10 10 4×10 10 5×10 10 6×10 10 7×10 10 8×10 10 Or 9×10 10 GC includes all integers or fractions within the range. In another embodiment, the composition is formulated to contain at least 1 × 10⁻⁶ per dose. 11 2×10 11 3×10 11 4×10 11 5×10 11 6×10 11 7×10 11 8×10 11 Or 9×10 11 GC includes all integers or fractions within the range. In another embodiment, the composition is formulated to contain at least 1 × 10⁻⁶ per dose. 12 2×10 12 3×10 12 4×10 12 5×10 12 6×10 12 7×10 12 8×10 12 Or 9×10 12 GC includes all integers or fractions within the range. In another embodiment, the composition is formulated to contain at least 1 × 10⁻⁶ per dose. 13 2×10 13 3×10 13 4×10 13 5×10 13 6×10 13 7×10 13 8×10 13 Or 9×10 13 GC includes all integers or fractions within the range. In another embodiment, the composition is formulated to contain at least 1 × 10⁻⁶ per dose. 14 2×10 14 3×10 14 4×10 14 5×10 14 6×10 14 7×10 14 8×10 14Or 9×10 14 GC includes all integers or fractions within the range. In another embodiment, the composition is formulated to contain at least 1 × 10⁻⁶ per dose. 15 2×10 15 3×10 15 4×10 15 5×10 15 6×10 15 7×10 15 8×10 15 Or 9×10 15 GC contains all integers or fractions within the range.

[0142] In one embodiment, for human application, the dosage range can be 1 × 10⁻⁶. 10 Up to approximately 1×10 15 GC per kg of body weight, encompassing all integers or fractions within the range. In one embodiment, the effective quantity of the carrier is approximately 1 × 10⁻⁶. 9 2×10 9 3×10 9 4×10 9 5×10 9 6×10 9 7×10 9 8×10 9 Or 9×10 9 GC per kg of body weight includes all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 10 2×10 10 3×10 10 4×10 10 5×10 10 6×10 10 7×10 10 8×10 10 Or 9×10 10 GC per kg of body weight includes all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 11 2×10 11 3×10 11 4×10 11 5×10 11 6×10 11 7×10 11 8×10 11 Or 9×10 11 GC per kg of body weight includes all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 12 2×1012 3×10 12 4×10 12 5×10 12 6×10 12 7×10 12 8×10 12 Or 9×10 12 GC per kg of body weight includes all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 13 2×10 13 3×10 13 4×10 13 5×10 13 6×10 13 7×10 13 8×10 13 Or 9×10 13 GC per kg of body weight includes all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 14 2×10 14 3×10 14 4×10 14 5×10 14 6×10 14 7×10 14 8×10 14 Or 9×10 14 GC per kg of body weight includes all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 15 2×10 15 3×10 15 4×10 15 5×10 15 6×10 15 7×10 15 8×10 15 Or 9×10 15 GC per kg of body weight, including all integers or fractions within the range.

[0143] In one embodiment, for human application, the dosage range can be 1 × 10⁻⁶. 10 Up to approximately 1×10 15 GC per gram (g) of brain mass, encompassing all integers or fractions within the range. In one embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 9 2×10 9 3×10 9 4×10 9 5×10 9 6×109 7×10 9 8×10 9 Or 9×10 9 GC per gram (g) of brain mass, encompassing all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 10 2×10 10 3×10 10 4×10 10 5×10 10 6×10 10 7×10 10 8×10 10 Or 9×10 10 GC per gram (g) of brain mass, encompassing all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 11 2×10 11 3×10 11 4×10 11 5×10 11 6×10 11 7×10 11 8×10 11 Or 9×10 11 GC per gram (g) of brain mass, encompassing all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 12 2×10 12 3×10 12 4×10 12 5×10 12 6×10 12 7×10 12 8×10 12 Or 9×10 12 GC per gram (g) of brain mass, encompassing all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 13 2×10 13 3×10 13 4×10 13 5×10 13 6×10 13 7×10 13 8×10 13 Or 9×10 13 GC per gram (g) of brain mass, encompassing all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 14 2×10 14 3×10 14 4×1014 5×10 14 6×10 14 7×10 14 8×10 14 Or 9×10 14 GC per gram (g) of brain mass, encompassing all integers or fractions within the range. In another embodiment, the effective amount of the carrier is approximately 1 × 10⁻⁶. 15 2×10 15 3×10 15 4×10 15 5×10 15 6×10 15 7×10 15 8×10 15 Or 9×10 15 GC refers to brain mass per gram (g), encompassing all integers or fractions within the range.

[0144] These dosages can be administered in various volumes of carrier, excipient, or buffer formulations, ranging from about 25 to about 1000 μL or more, encompassing all numbers within that range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume of the carrier, excipient, or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 225 μL. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In another embodiment, the volume is approximately 350 μL. In another embodiment, the volume is approximately 375 μL. In another embodiment, the volume is approximately 400 μL. In another embodiment, the volume is approximately 450 μL. In another embodiment, the volume is approximately 500 μL. In another embodiment, the volume is approximately 550 μL. In another embodiment, the volume is approximately 600 μL. In another embodiment, the volume is approximately 650 μL. In another embodiment, the volume is approximately 700 μL. In another embodiment, the volume is between approximately 700 and approximately 1000 μL.

[0145] In some embodiments, the dose can be approximately 1 × 10⁻⁶. 9 GC / g brain mass up to approximately 1×1012 Within the range of GC / g brain mass. In some embodiments, the dose may be approximately 1 × 10⁻⁶. 10 GC / g brain mass up to approximately 3×10 11 Within the range of GC / g brain mass. In some embodiments, the dose may be approximately 1 × 10⁻⁶. 10 GC / g brain mass up to approximately 2.5 × 10⁻⁶ 11 Within the range of GC / g brain mass. In some embodiments, the dose may be approximately 5 × 10⁻⁶. 10 Within the range of GC / g brain mass.

[0146] In one embodiment, the viral construct can be at least about 1×10 9 GC up to approximately 1×10 15 Or approximately 1×10 11 Up to 5×10 13 Dosage delivery of GC. The appropriate volume for delivering these doses and concentrations can be determined by those skilled in the art. For example, volumes from about 1 μL to 150 mL can be selected, with larger volumes chosen for adults. Typically, for newborns, appropriate volumes are from about 0.5 mL to about 10 mL, and for older infants, from about 0.5 mL to about 15 mL. For young children, volumes from about 0.5 mL to about 20 mL can be selected. For children, volumes up to about 30 mL can be selected. For pre-pubertal adolescents and teenagers, volumes up to about 50 mL can be selected. In other embodiments, patients may receive intrathecal administration volumes selected from about 5 mL to about 15 mL or from about 7.5 mL to about 10 mL. Other appropriate volumes and doses can be determined. The dose can be adjusted to balance therapeutic benefits with any side effects, and this dose can vary depending on the therapeutic application employing the recombinant carrier.

[0147] The recombinant vector described above can be delivered to host cells according to the disclosed methods. rAAV, preferably suspended in a physiologically compatible carrier, can be administered to human or non-human mammalian patients. In some embodiments, for administration to human patients, rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Since the pH of cerebrospinal fluid is about 7.28 to about 7.32, a pH within this range may be desired for intrathecal delivery; while for intravenous delivery, a pH of about 6.8 to about 7.2 may be desired. However, other pH ranges and subranges thereof can be selected for other delivery routes.

[0148] As used herein, the terms "intrathecal delivery" or "intrathecal administration" refer to a route of drug administration that delivers the drug into the cerebrospinal fluid (CSF) by injection into the spinal canal, more specifically into the subarachnoid space. Intrathecal delivery can include lumbar puncture, intracardiac (including intraventricular (ICV)), suboccipital / cisternal, and / or C1-2 puncture. For example, material can be introduced via lumbar puncture to diffuse throughout the subarachnoid space. In another instance, it can be injected into the cerebellomedullary cistern. In some embodiments, rAAV, carriers, or compositions as described herein are administered to a subject in need via intrathecal administration. In some embodiments, intrathecal administration is performed as described in U.S. Patent Publication No. 2018-0339065A1, published November 29, 2019, which is incorporated herein by reference in its entirety.

[0149] As used herein, the terms "intracisional delivery" or "intracisional administration" refer to the direct entry of a drug into the cerebrospinal fluid of the cerebellomedullary cistern, more specifically via suboccipital puncture or by direct injection into the cerebellomedullary cistern or by a permanently positioned tube.

[0150] In some embodiments, treatment with the compositions described herein induced minimal to mild asymptomatic degeneration of DRG sensory neurons in animal and / or human patients and was well tolerated in terms of sensory neurotoxicity and subclinical sensory neuropathic effects.

[0151] VII. Apparatus and methods for delivering a pharmaceutical composition into cerebrospinal fluid.

[0152] On the one hand, the carriers provided herein can be administered intrathecally using the methods and / or devices provided in this section and described in WO 2018 / 160582, which is incorporated herein by reference. Alternatively, other devices and methods may be chosen. In some embodiments, the method includes the step of suboccipital injection via a spinal needle guided by CT into the cerebellomedullary cistern of a patient. As used herein, the term computed tomography (CT) refers to radiography in which a three-dimensional image of a body structure is constructed by a computer from a series of planar cross-sectional images made along an axis. In some embodiments, the device is described in U.S. Patent Publication No. 2018-0339065 A1, published November 29, 2019, which is incorporated herein by reference in its entirety.

[0153] VIII.CDD

[0154] In this document, the terms "patient" or "subject" may be used interchangeably to mean a male or female mammal, including humans, veterinary or farm animals, livestock or pets, and animals commonly used in clinical research. In one embodiment, the subject of these methods and compositions is a human patient. In one embodiment, the subject of these methods and compositions is a male or female human. In some embodiments, the subject of these methods and compositions is diagnosed with CDD and / or has symptoms of CDD.

[0155] The methods and compositions can be used to treat any of the following stages of CDD. In some embodiments, patients are approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months old, or approximately 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, 10.5 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, or 18 years old. In some embodiments, patients are infants, for example, 18 months to 3 years old. In some embodiments, patients are 3 to 6 years old, 3 to 12 years old, 3 to 18 years old, or 3 to 30 years old. In some embodiments, patients are 18 years of age or older.

[0156] Symptoms of CDD include seizures that typically begin within the first three months of life, but can appear as early as the first week after birth. The type of seizure varies with age and may follow a predictable pattern. The most common types are generalized tonic-clonic seizures, which involve loss of consciousness, muscle stiffness, and convulsions; tonic seizures characterized by abnormal muscle contractions; and epileptic spasms, which involve brief episodes of muscle twitching. Most people with CDKL5 deficiency experience seizures daily, but they may have periods without seizures. Seizures in CDKL5 deficiency are often resistant to treatment.

[0157] Children with CDKL5 deficiency experience developmental impairments. Most have severe intellectual disabilities and little or no verbal ability. The development of gross motor skills, such as sitting, standing, and walking, is delayed or impossible. About one-third of affected individuals are able to walk independently. Fine motor skills (such as picking up small objects with fingers) are also impaired; about half of affected individuals use their hands purposefully. Most people with this condition have visual problems (cortical visual impairment).

[0158] Other common features of CDKL5 deficiency include repetitive hand movements (stereotypes), such as clapping, licking, and sucking; teeth grinding (bruxism); sleep disturbances; feeding difficulties; and gastrointestinal problems, including constipation and acid reflux into the esophagus (gastroesophageal reflux). Some affected individuals experience episodes of irregular breathing. Distinctive facial features of some people with CDKL5 deficiency include a high and broad forehead, large and deep-set eyes, a well-defined space between the nose and upper lip (philtrum), full lips, widely spaced teeth, and a high roof of the mouth (palate). Other physical differences may also be present, such as an abnormally small head size (microcephaly), scoliosis (bending of the spine to the left or right), and tapering fingers.

[0159] As described above, unless otherwise specified, the terms “increase,” “decrease,” “reduce,” “alleviate,” “improve,” “delay,” or any similar terms of any grammatical variation or indicative change thereof mean approximately 5-fold, approximately 2-fold, approximately 1-fold, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 10%, or approximately 5% of the corresponding reference (e.g., an untreated control or a subject in a normal condition without CDD).

[0160] In some embodiments, patients receive medication to control some signs and symptoms associated with CDD, such as seizures, muscle stiffness or breathing, sleep, gastrointestinal or cardiac problems.

[0161] In some embodiments, a diuretic may be used as a co-treatment for subjects in need. The diuretic used may be acetazolamide (Diamox) or other suitable diuretics. In some embodiments, the diuretic is administered at the same time as gene therapy. In some embodiments, the diuretic is administered before gene therapy. In some embodiments, the diuretic is administered in an injection volume of 3 mL.

[0162] In some embodiments, co-therapy may be utilized, which includes the combined administration of Cdkl5-isotype 1, isotype 2, isotype 3 and / or isotype 4 expression vectors or various bidirectional or tridirectional combinations thereof. Optionally, co-therapy may further include the administration of another active agent. In some embodiments, co-therapy may include enzyme replacement therapy.

[0163] Optionally, immunosuppressive co-therapy may be used on subjects who require it. Immunosuppressants used in such co-therapy include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapamycin analogs), and cell growth inhibitors, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunoaffinities. Immunosuppressants may include nitrogen mustard, nitrosourea, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25) or CD3-targeted antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-α) binders. In some embodiments, immunosuppressive therapy may be initiated on days 0, 1, 2, 3, 4, 5, 6, 7, or more before or after gene therapy administration. Such immunosuppressive therapies may involve the administration of one, two, or more drugs (e.g., glucocorticoids, prednisone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)). These immunosuppressive drugs may be administered once, twice, or multiple times to the subject in need at the same or adjusted dose. Such therapies may involve the co-administration of two or more drugs (e.g., prednisone, mycophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued at the same or adjusted dose following gene therapy administration. Such therapies may continue for approximately one week (7 days), approximately 60 days, or longer, as needed. In some embodiments, a tacrolimus-free regimen is selected.

[0164] The terms “comprise,” “comprises,” and “comprising” will be interpreted as inclusive rather than exclusive. The terms “consist,” “consisting,” and variations thereof will be interpreted as exclusive rather than inclusive. While various embodiments in the specification are presented using the language of “comprises,” in other instances, the language of “consisting” or “substantially constituting” is intended to explain and describe related embodiments.

[0165] The term "expression" is used in its broadest sense herein and includes the production of RNA or RNA and proteins. Regarding RNA, the term "expression" or "translation" specifically refers to the production of peptides or proteins. Expression can be transient or stable.

[0166] It should be noted that the term "a / an" refers to one or more, for example, "an enhancer" should be understood to mean one or more enhancers. Thus, the terms "a" (or "an"), "one or more", and "at least one" are used interchangeably in this document.

[0167] Throughout the instruction manual, the term "e" followed by a numerical value (n) is used to refer to the exponent. It should be understood that this refers to "×10⁻¹⁰". n For example, "3e9" is different from 3×10. 9 The same, and "1e13" is the same as 1×10 13 same.

[0168] As described above, unless otherwise stated, the term “about” when used to modify numerical values ​​means a change of ±10%.

[0169] Unless otherwise defined in this specification, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art and by reference to the published text, which provides a general guide for those skilled in the art to the many terms used in this application.

[0170] IX. Instance

[0171] The following examples are merely illustrative and are not intended to limit the invention.

[0172] Several CDD models have been developed and can be selected for evaluating treatment efficacy. The following models are ineffective for CDKL5 expression: for example, Cdkl5-ko mice with deletions in exon 6 (Δ exon 6), Wang et al., Proceedings of the National Academy of Sciences, Dec. 2012, 109(52) 21516-21521; DOI: 10.1073 / pnas.1216988110; Cdkl5-ko mice with deletions in exon 4 (Δ exon 4) (see Amendola et al. (2014), Mapping Pathological Phenotypes in a Mouse Model of CDKL5Disorder, PLOS ONE). ONE)》9(5):e91613.doi.org / 10.1371 / journal.pone.0091613(2014); Cdkl5(R59X knock-in) model (available from Jackson Laboratory; see Tang et al. (2019), Tang S et al. altered NMDAR signaling underlies autistic-like features in mouse models of CDKL5 deficiency disorder). Nature Communications (NatCommun.) 2019; 10(1):2655. Published on June 14, 2019. doi:10.1038 / s41467-019-10689-w; and Cdkl5(D471fs) model (Rodney Samaco, Baylor College of Medicine, Houston, TX.

[0173] This study demonstrated that restoring CDKL5 expression in the CNS of three CDD mouse models (Cdkl5-ko (exon 6), Cdkl5 (R59X), and Cdkl5 (D471fs)) significantly improved disease symptoms. An AAV gene therapy vector was developed comprising an AAVhu68 capsid, an expression cassette with a human synaptic protein promoter, and an engineered human CDKL5 transgene. When the AAV-CDKL5 vector was administered to Cdkl5 knockout mice via neonatal injection into the lateral ventricle, strong expression was detected in up to 50% of neurons throughout the brain. AAV administration and human CDKL5 expression were well tolerated. Human CDKL5 was primarily localized in the cytoplasm; protein expression and kinase activity persisted for more than 4 months. A series of neurobehavioral tests were performed on the treated Cdkl5-ko mouse cohort, and the untreated Cdkl5-ko phenotype was found to significantly improve behavioral outcomes observed in wild-type mice. The same study was then repeated in two different CDD mouse models carrying patient-derived frameshift mutations (Cdkl5(R59X) and CDKL5(D471fs) models) rather than gene knockout. Very similar results were obtained, thus reaffirming the curative benefits of CDKL5 gene therapy vectors.

[0174] CDKL5 is expressed in the human brain in at least four different isoforms. Similar AAV gene therapy vectors were generated for three alternative splicing isoforms, all of which were found to be expressed in the transduced mouse brains and exhibit kinase activity.

[0175] To test the expression of the AAV-CDKL5 gene therapy vector in larger animals, a study was conducted using rhesus monkeys. By injecting cerebrospinal fluid via the cerebellomedullary cistern, the vector was distributed throughout the brain at 0.1 to 1 copy per diploid genome. Much higher vector transduction was observed in the dorsal root ganglia (DRG). Therefore, in situ hybridization with probes specific to the human CDKL5 sequence showed abundant expression in DRG neurons, but much sparser expression in cortical gray matter neurons. CDKL5 administration and expression were generally well tolerated in all six rhesus monkeys for 60 days; however, mild axonal lesions in the spinal white matter tracts were noted.

[0176] In summary, this study demonstrates promising evidence that CDKL5 gene therapy provides durable therapeutic benefits in model mice and is well-tolerated in non-human primates. Further optimization of this approach may ultimately provide a clinical intervention option for children affected by CDD.

[0177] Example 1. Materials and Methods.

[0178] Plasmids in the human brain 1The amino acid sequences of four CDKL5 (cyclin-dependent kinase-like 5, Uniprot IDO76039) expressed in the genome were reverse-translated into DNA sequences. The coding sequences were further engineered, for example, by taking into account codon frequencies found in the human genome, hidden RNA splicing sites, and alternative reading frames. The engineered sequences were cloned into AAV expression plasmids under the control of the human synaptic protein promoter. 2 The coding sequence is preceded by a Kozak sequence, followed by a WPRE enhancer cassette (a posttranscriptional regulatory element of marmot hepatitis virus), an SV40 poly A sequence, and framed by an AAV2 inverted terminal repeat (ITR). To suppress expression in the dorsal root ganglion (DRG), in some experiments, the plasmid was modified to contain four repeat sequences directly after the CDKL5 coding sequence and before the WPRE sequence, containing the miR183 binding site (agtgaattctaccagtgccata) (SEQ ID NO:11). The AAV CDKL5 vector was generated at Vector Core, University of Pennsylvania, as a capsid PHP.B. 3 For use in mouse studies, or as AAV9hu68 4 AAVrh91 can be used for research in mice and non-human primates.

[0179] Mouse Studies. All studies involving mice were approved by the University of Pennsylvania IACUC. Cdkl5-ko mice (B6.129(FVB)-Cdkl5tm1.1Joez / J, strain #021967) were obtained from the Jackson Laboratory, and heterozygous female ko mice were crossed with wt C57Bl6 males to obtain the following littermate genotypes: male hemizygous Cdkl5-ko (also known as KO mice or wt mice), male wt, female heterozygous Cdkl5-ko, and female wt. Mice received 100 μl of AAV Cdkl5 vector (dose range 1 × 10⁻⁶) via retroorbital injection at 18–21 days of age. 11 GC up to 5×10 11 GC) or mediator control (sterile phosphate-buffered saline). Alternatively, mice were administered an intracerebrospinal injection on the day of birth at a dose of 1 × 10⁻⁶. 10 GC up to 5×10 10 GC, total volume 2 μl. For both genotype and injectable product, mice were housed in mixed groups, weighed and observed at least twice weekly, and males were 11 weeks old or females were 14 weeks old when subjected to behavioral testing. No treatment-related morbidity was observed.

[0180] Western blotting and tissue staining. Following euthanasia, one cortical hemisphere was rapidly frozen, and protein lysates were subsequently generated using RIPA buffer. Western blotting was performed using antibodies against human CdKL5 (S957D, University of Dundee, UK), EB2 (ab45767, Abcam), or EB2 phospo222 (pab01032-P, Covalab, UK). The other hemisphere of the brain was fixed overnight in formalin, embedded in paraffin, and thin sections were processed for immunofluorescence staining with the same CDKL5 antibody.

[0181] Behavioral Testing. Mice were tested once daily. The testing time, operator, and environment remained consistent each day (60 dB white noise background and 1000 lumens incandescent indirect illumination). Before each test, mice were acclimatized to their living cages for 30 minutes. For open field assays, new cages with minimal bedding were placed in an infrared beam array (MedAssociates, Inc.). A mouse was added to the center of the cage, and the number of beam interruptions over the next 30 minutes was automatically recorded, categorized as beam interruptions near the ground (normal activity) and beam interruptions 3 inches above the ground (upright activity). For the Elevated Zero Maze (EZM, Stoelting Co.), an elevated circular platform with two relatively closed quadrants and two open quadrants was used to allow uninterrupted exploration. A single mouse was placed in the center of the open quadrant, and movement was video-recorded for 15 minutes. For the Y Maze (Stoelting), a Y-shaped closed platform with three identical arms was used. A single mouse was placed in the arm closest to the operator, and its movements were video-recorded for 5 minutes. For bead embedding assays, a new cage was filled with 3 inches of AlphaDri bedding (Shepherd Specialty Papers) and gently compacted. Twelve pure blue beads were evenly spaced on the bedding, and a single mouse was placed in the center of the cage. After 30 minutes, the number of beads that were at least half covered by the bedding was recorded.

[0182] Data Analysis. Data were plotted and analyzed using GraphPad Prims software. Video files were recorded in mp4 format at 20fps and analyzed using EthoVision XT software (version 14, Noldus Information Technology). For nesting assays, mice were individually housed overnight in new cages supplied with standardized 2×2-inch nestlet blocks (cotton-based). After 24 hours, nest quality was scored on a scale of 1-5, and any remaining untouched nestlet material was weighed.

[0183] Non-human primate experiments. All studies involving non-human primates were approved by the University of Pennsylvania IACUC and conducted in accordance with USDA regulations. Non-human primates (NHPs) of the rhesus macaque species were obtained from Covance Research Products, Inc. Quarantine and domestication were conducted according to Gene Therapy Program SOPs. Body weight, temperature, respiratory rate, and heart rate were monitored regularly one month prior to AAV vector administration and throughout the study, and blood and CSF samples were obtained. Whole blood was used for cell counting and differential analysis, as well as clinical blood chemistry panel analysis. CSF samples were used for blood cell counting and differential analysis, and total protein quantification. To deliver the AAV vector into the CSF via puncture of the cerebellomedullary cistern, anesthetized rhesus macaques were placed on the operating table in a lateral decubitus position with their heads bent forward. Using aseptic technique, a 21-27 gauge, 1-1.5 inch Quincke spinal needle (Becton Dickinson) is advanced into the suboccipital space until CSF flow is observed. The needle is directed toward the wider superior space of the cerebellomedullary cistern to avoid blood contamination and potential brainstem injury. Correct needle placement is verified by myelography using a fluorescence microscope. 1 mL of CSF is collected for baseline analysis prior to administration. After CSF collection, a Leur pathway extension catheter is connected to the spinal needle to facilitate administration of 1 mL of iohexol (trade name: Onexol 180 mg / mL, General Electric Healthcare). After verifying needle placement, a syringe containing the test product (volume equal to 1 mL plus the volume of the syringe and connector dead zone) is connected to the flexible connector and injected for 30 ± 5 seconds. The needle is removed and pressure is applied directly to the puncture site. The AAVhu68.hSyn.Cdkl5-1co and AAVhu68.hSyn.Cdkl5-1co carriers have a maximum capacity of 3 × 10⁻⁶. 13GC / NHP dosing was administered. Neurological assessments were performed on all macaques on days 0, 14, 18, 41, and the final study day to evaluate neurological function in detail. Briefly, the assessments included postural and gait assessments, cranial nerve assessments, proprioceptive assessments, and spinal / neural reflexes. On study day 56, the macaques were euthanized, and total necropsy and autopsy were performed. Twenty-five major tissue samples were harvested from each macaque in duplicate for rapid freezing or fixation in formalin. DNA or RNA was purified from the rapidly frozen tissues and used for vector biodistribution or transgene expression analysis, respectively. For vector biodistribution, TaqMan qPCR assays were used to determine the genomic copy number (GC) per total DNA weight, with the probe again pointing to the polyA region of the transgene cassette and an internal standard. To quantify transgene expression, total RNA was used to generate cDNA via first-strand synthesis with polyT oligonucleotides, followed by TaqMan qPCR using probes specific for transgenes that do not cross-react with the endogenous rhesus macaque CDKL5 sequence. For histopathological analysis, macaque tissue was embedded in paraffin, and these sections were stained with either H&E solution or CDKL5 antibody. Identical spinal cord sections were incubated with Lauker's Fast Blue to stain the myelin sheath. All stained tissue sections were reviewed by a professionally qualified veterinary pathologist, and any abnormalities were peer-verified.

[0184] References:

[0185] 1. Hector, RD et al. Characterization of CDKL5 Transcript Isoforms in Human and Mouse. PLOS ONE 11, e0157758, (2016).

[0186] 2. Thiel, G., Greengard, P. & Sudhof, TC. Characterization of tissue-specific transcription by the humansynapsin I gene promoter. Proceedings of the National Academy of Sciences of the United States of America 88, 3431-3435, (1991).

[0187] 3. Deverman, BE et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nature Biotechnol 34, 204-209, (2016).

[0188] 4. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Human Gene Therapy 29, 285-298, (2018).

[0189] Example 2. Gene therapy AAV vector

[0190] The expression construct between ITRs contains the human synaptic protein promoter (SEQ ID NO:23), an engineered coding sequence of human CDKL5 isotype 1 (SEQ ID NO:22), the WPRE expression enhancer (SEQ ID NO:27), and the SV40 poly A sequence (SEQ ID NO:28). Figure 1A Similarly, alternative expression constructs contained human CDKL5, isotype 2 (CDKL5-2GS or hCDKL5-2GS; SEQ ID NO:24), 3 (CDKL5-3GS or hCDKL5-3GS; SEQ ID NO:25), or 4 (CDKL5-4GS or hCDKL5-4GS; SEQ ID NO:26), respectively, instead of the engineered coding sequence of isotype 1. All tested plasmids expressed well in mouse brains, showing minor differences in the ability to phosphorylate EB2 (measurement of CDKL5 kinase activity). It was found that the WPRE enhancer was required to obtain human Cdkl5 expression levels in mouse brains similar to wild-type mouse CDKL5 expression (Figure 2). CDKL5 was fully active, as determined by the ability of said CDKL5 to phosphorylate its endogenous target protein EB2 (Figure 2). The expression and localization of CDKL5 were confirmed by IHC. The expression and localization of CDKL5 were also confirmed by the use of human synaptic protein with the human ubiquitin C promoter (Ubc). Figure 1B) or chicken β-actin heterozygous promoter ( Figure 1C Exchange to generate alternative expression constructs. Neonatal ICV in Cdkl5-ko mice was 3 × 10⁻⁶. 10 GC was administered AAV vectors AAVrh91.UbC.CDKL5-1co.miR183 and AAVrh91.CBh.CDKL5-1co.miR183 from the AAV capsid, and necropsy was performed at P14. Western blot analysis of mouse brain tissue confirmed CDKL5 expression after transduction with AAV vectors including either the UbC or CBh promoter. CDKL5 transgene expression was observed in the mouse brain after transduction with AAVrh91.UbC.CDKL5-1co.miR183 and AAVrh91.CBh.CDKL5-1co.miR183. Higher per-cell CDKL5 expression was observed in the hippocampus of the mouse brain after transduction with AAVrh91.UbC.CDKL5-1co.miR183. In contrast, after transduction with AAVrh91.CBh.CDKL5-1co.miR183, lower CDKL5 expression per cell was observed in the hippocampus of mice. After transduction with AAVrh91.UbC.CDKL5-1co.miR183, higher CDKL5 expression per cell was observed in the cerebral cortex of mice. After transduction with AAVrh91.CBh.CDKL5-1co.miR183, lower CDKL5 expression per cell was observed in the cerebral cortex of mice. Furthermore, the expression of CDKL5 was compared between cells treated with AAVrh91.UbC.CDKL5-1co.miR183 (3×10⁻⁶). 10 GC, neonatal ICV) and AAVhu68.hSyn.CDKL5-1co.miR183 (2.5×10 10 CDKL5 expression in the brains of Cdkl5-ko mice after GC (Genetic Grafting). A very similar expression pattern was observed in the mouse brains after administration of AAV as directed. CDKL5 expression was observed in the mouse brains after transduction with AAVrh91.UbC.CDKL5-1co.miR183 (AAVrh91 capsid; ubiquitin C promoter) or with AAVhu68.hSyn.CDKL5-1co.miR183 (AAVhu68 capsid; synaptic protein promoter). Additionally, CDKL5 expression was examined in Cdkl5-ko mice at a concentration of 1×10⁻⁶. 10 GC, 3×10 10 GC and 6×10 10 Behavioral effects of different doses of GC on AAVrh91.UbC.CDKL5-1co.miR183 and AAVrh91.CBh.CDKL5-1co.miR183.

[0191] The AAV.UbC.CDKL5-1co.miR183 vector exhibited therapeutic efficacy in mice similar to that when CDKL5 expression is driven by hSyn. When used with the AAV vector genome including engineered nucleic acid sequences, the AAVrh91 capsid achieved CDKL5 expression similar to that of AAVhu68. The Ubc promoter resulted in higher CDKL5 protein levels in the mouse brain compared to the hSyn promoter.

[0192] Example 3. Preclinical therapeutic benefits of CDKL5 gene therapy in CDD mouse models

[0193] To test the therapeutic benefit of CDKL5 gene therapy in Cdkl5-deficient mice, 5 × 10⁵ CDKL5 gene therapy was administered via retroorbital IV injection. 11 The GC(5e11 gc)AAV9-PHP.B-hSyn-hCDKL5-1co.WPRE vector was used to treat cohorts of juvenile Cdkl5-ko (also known as KO mice or slugs) and wild-type (wt) littermates. All treatment groups continued to increase at the same rate, and no treatment-related deaths were observed. At 10 weeks of age, the mice were subjected to a set of behavioral tests. Robust and statistically significant normalization was observed in the elevated zero maze and open field activity tests in the treatment groups. Minor improvements were observed in the robin and Y-maze tests in the same groups, but no improvement was observed in thermal sensitivity.

[0194] Given the importance of Cdkl5 for neural development, we wanted to know if early administration of gene therapy could improve treatment outcomes in mice. Next, we administered 6 × 10⁶ Cdkl5 per mouse via intracardiac transventricular (ICV) injection. 9 GC up to 5×10 10 A cohort of newborn Cdkl5-ko or wt littermates were treated with AAVhu68-hSyn-hCDKL5-1co.WPRE vectors within the GC dose range. Behavioral testing was performed at 10 weeks of age or 11–14 weeks of age as instructed. Overall observations across all groups showed good treatment tolerance, no treatment-related deaths, and normal weight gain. Figure 9A and 9B ) and overall development. At 5×10 10 The dose of GC, the dose of 2.5 × 10¹⁰ GC, and the dose of 1 × 10¹⁰ GC. 10At GC doses, dose-dependent expression of the CDKL5 transgene in the brains of Cdkl5-ko mice was observed. At 10 weeks of age, KO mice exhibited a characteristic hindlimb clenching phenotype, which was significantly improved in treated KO mice. The therapeutic efficacy of CDKL5 gene therapy was measured by a hindlimb clenching test. Dose-dependent improvements with severity scores were observed in treated CDKL5-ko mice. Figure 9C-9F Similarly, the persistent hyperactivity and fed phenotype observed in KO mice were normalized to wild-type activity. In the Y-maze test (spontaneous alteration index), hippocampal learning and memory were significantly improved in treated KO mice. The therapeutic efficacy of CDKL5 gene therapy was further measured by the open field activity test, which measures the correction for hyperactivity in KO and AAV-treated mice. A dose-dependent reduction in hyperactivity was observed in treated Cdkl5-KO mice. Figure 11A-11F KO mice, when housed alone overnight in new cages, are not adept at nest building and do not destroy all nesting material. Treatment of KO mice significantly improved nesting ability in a dose-dependent manner. Figure 10A , 10C 10E). Two other neurobehavioral assays (bead embedding and Y-maze) showed a strong trend toward phenotypic improvement in treated Cdkl5-ko mice. Figure 10B and 10D The EEG phenotype was rescued in the initial collaborative study and may serve as a translational biomarker. Validation of key assays (hindlimb clenching, gait activity) was performed additionally using independent experimental cohorts. Larger cohort sizes (N=18 / group) resulted in more robust statistical significance. Validation of key assays (hindlimb clenching, gait activity) was also performed using alternative CDD mouse models (R59X, D471fs). In summary, CDKL5 gene therapy delivers functional CDKL5 protein to the mouse brain and demonstrates dose-dependent therapeutic effects on several neurobehavioral outcomes in a CDD male mouse model.

[0195] After completing behavioral tests, brain samples were collected from mice at approximately 3 months of age. Western blotting revealed strong expression of human CDKL5 in the Cdkl5-ko brain, even 3 months after AAV administration. Finally, human CDKL5 exhibited robust kinase activity when blotted against phosphorylated EB2 protein.

[0196] The effects of CDKL5 gene therapy were also tested using an alternative CDD mouse model. Cdkl5(D471fs) carried a patient-associated point mutation leading to a premature stop codon. Similar to patients, CDKL5 protein and highly reduced EB phosphorylation levels were not found in the brains of these mice. Therefore, Cdkl5(D471fs) lacked Cdkl5 protein, as previously used in Cdkl5-ko mice; however, the genetic background differed slightly due to the method used to generate the mouse model. Newborn mice were injected with the same concentration as before (5 × 10⁻⁶). 10 GC (neoICV) and showed robust hCDKL5 protein expression and EB2 phosphorylation after 3 months. A small experimental cohort was subjected to behavioral tests. AAV treatment was well tolerated and no morbidity was observed. Hind limb clasping was significantly corrected in treated mutant mice; similarly, the mutant phenotype in the elevated zero maze test was corrected to wild-type behavior (located in open areas, number of times open areas were entered) in treated mutant mice. In new cages, mutant mice showed poor interaction with glass beads arranged in a grid and burying behavior, while wild-type mice typically buried almost all glass beads. Treated mutant mice showed strong correction for the behavior of wild-type mice. 10 At higher doses of GC / mouse, significant improvements were observed in Cdkl5-ko mice in the treatment group.

[0197] The effects of CDKL5 gene therapy were also tested using an alternative CDD mouse model. Cdkl5(R59X) carried a patient point mutation that introduced an early stop codon. Similar to patients, CDKL5 protein and highly reduced EB phosphorylation levels were not found in the brains of these mice. Therefore, Cdkl5(R59X) lacks Cdkl5 protein, as previously used in Cdkl5-ko mice; however, the genetic background is slightly different due to the method used to generate the mouse model. Newborn pups were injected with the same concentration as before (5 × 10⁻⁶). 10 GC (neoICV) and showed robust hCDKL5 protein expression and EB2 phosphorylation after 3 months. A small experimental cohort was subjected to behavioral tests. AAV treatment was well tolerated and no morbidity was observed. Hind limb tethering was significantly corrected in the treated mutant mice.

[0198] The outcomes of CDKL5 gene therapy were also examined using an alternative CDD mouse model in heterozygous female Cdkl5-ko mice. This model reflects the majority of CDD patients (CDD females). Overall assessment showed that the neurobehavioral phenotype of heterozygous female Cdkl5-ko mice was much milder, with a later onset of disease, making reliable assessment of treatment outcomes more difficult. However, at high doses (5 × 10⁻⁶), the outcomes were better assessed. 10Representative data from GC (neonatal ICV) showed significant improvements in hind limb clenching and walking activity. Figure 14B and 14C ).

[0199] Furthermore, dose escalation of CDKL5 gene therapy was detected in WT mice. WT (C57Bl6 / J) mice were treated with neonatal ICV at a dose of 7.5 × 10⁻⁶. 10 GC and 1×10 11 GC (i.e., 1.5 or 2 times the previously highest administered dose) was administered. No significant effects on body weight, development, or survival were observed. Mice behaved normally and showed no changes in hind limb clenching or movement. No effects were observed in pathological examination of CNS tissues. Figure 19A and 19B ).

[0200] Example 4. Practitioner CDKL5 type 2-4

[0201] At least four detectable Cdkl5 mRNA splice variants have been shown to exist in the human and mouse brains, but isotypes 2–4 have not yet been identified as stable proteins. Isotype 1 accounts for more than 85% of brain CDKL5.

[0202] The coding sequences for human CDKL4 isoforms 2-4 were codon-engineered and cloned into the same AAV vector as previously used. The AAV9-PHP.B vector encoding isoforms 1-4 was tail-IV-injected into adult Cdkl5-ko mice, and brain samples were collected after 2 weeks for Western blot analysis. All four isoforms were found to be robustly expressed and exhibit the expected gel migration pattern. Engineered sequences producing expression levels of isoforms 2-4 very similar to isoform 1 were selected for follow-up. Isoform 2 produced slightly higher EB2 phosphorylation levels compared to isoform 1, while isoforms 3 or 4 produced slightly lower EB2 phosphorylation levels.

[0203] Three alternative CDKL5 isoforms have been injected into newborn Cdkl5-ko mice to test their potential therapeutic benefits compared to isoform 1. Figure 12 Significant correction of the fastening phenotype was demonstrated by treatment with alternative CDKL5 isotypes (2, 3 and 4). Figures 8A to 8D It provides the CDKL5 expression level or activity for AAV.CDLK5 vector constructs expressing isotype 1, isotype 2, isotype 3, or isotype 4. Figure 8A The results show the difference between wild-type mice injected with the causative agent and knockout mice injected with the causative agent, and mice injected with the causative agent that were injected with AAV vectors expressing each of these isotypes (5 × 10⁻⁶). 10 Expression levels of GC (newborn ICV) knockout mice. Figure 8B The CDKL5 activity was demonstrated as determined by pS222EB2 in wild-type mice injected with medulant (PBS), knockout mice injected with medulant, or AAV.CDKL5-1co. Figure 8C This shows the effect of using pS222EB2 on... Figure 8A The CDKL5 activity was measured in the group described in the study. Figure 8D It shows Figure 8B The CDKL5 expression level of the group described in the article.

[0204] In summary, all isotypes are expressed as proteins and possess considerable catalytic activity. When tested head-to-head (5 × 10⁻⁶), 10 In GC (neonatal ICV), treatment outcomes with isotypes 2, 3, or 4 in the CDD mouse model were comparable to those with isotype 1. Overall, CDKL5 gene therapy with CDKL5 isotype 1 is a promising and safe approach with significant therapeutic benefits in male CDD mouse models.

[0205] Example 5. Preliminary NHP study on the toxicity and safety of hCDKL5 gene therapy.

[0206] The aim was to investigate the expression patterns and safety characteristics of the AAV-hSyn-CDKL5-1co.WPRE construct encapsulated in an AAVhu68 capsid within an NHP. The vector was generated using the vector genome described herein and the previously described generation method. See, for example, WO 2018 / 160582, which is incorporated herein by reference. A group of six rhesus macaques (aged 4–6 years) were injected via the cerebellomedullary cistern (ICM). The vector was administered using the diuretic acetazolamide. After pretreatment for 2 days to reduce CSF generation, different conditions were tested:

[0207] A. Dosage 1×10 14 GC, injected into 1 ml buffer.

[0208] B. Dosage 1×10 14 GC, injected into 3ml or 5ml buffer.

[0209] C. Dosage 3×10 14 GC, injected into 3 ml buffer.

[0210] D. Dosage 1×10 14 GC was injected into 1 ml of buffer.

[0211] In summary, for non-human primate (NHP) studies, the AAVhu68-hSyn-Cdkl5-1co-WPRE vector was used to perform toxicity and safety testing of hCDKL5 gene therapy. In the preliminary study, three different doses were evaluated: 3 × 10⁻⁶. 12 GC / Animal, 1×10 13 GC / animals and 3×10 13 GC / animals. In the preliminary study, 1×10⁻⁶ was selected. 14 The dosage of GC / animals was evaluated, and the delivery of two different volumes (3 mL and 5 mL) of AAV carrier to cerebrospinal fluid (CSF) via the cerebellomedullary cistern was assessed. Other research groups used 1×10 14 GC / animals and diuretics (e.g., Danmus brand acetazolamide) or 3×10 14 GC / Animal (Subject).

[0212] In addition, 2×10 were tested. 12 1×10 13 and 3×10 13 GC / subject dosage. Treatment with the CDKL5 vector was well tolerated, and no signs of clinical changes in blood chemistry were observed. Neurological examination of the cage side revealed no changes compared to baseline.

[0213] Autopsy was performed 28 days after injection, followed by molecular analysis, histological, and pathological review. Overall, no major transduction differences were observed in major organs outside the CNS (e.g., liver transduction may have already reached its maximum). No major transduction differences were observed in the spinal cord and DRG (very high transduction rates were maintained), but significant changes dependent on injection parameters became apparent in brain tissue. The highest transduction increases were seen in the cortex. Danmus resulted in a slight increase in transduction efficiency across the brain.

[0214] Figure 17 The carrier biodistribution of each NHP in non-neuronal tissues, spinal tract tissues, and brain tissue is shown. Figure 18Only data on the carrier biodistribution in the brain are shown. Results indicate strong transduction in the dorsal root ganglion (DRG), moderate to low transduction in brain tissue, and transduction leakage into neuronal tissue. Pathological findings suggest mild axonal lesions in the dorsal white matter tract. Slight differences in hCDKL5 mRNA levels were observed. A trend toward higher mRNA expression was observed when using a 3 ml injection volume. hCDKL5 mRNA distribution was also visualized by in situ hybridization (ISH). The DRG showed very strong mRNA expression in all six NHPs. A small number of transducing neurons (<10%) were observed in the motor cortex. Small clusters of transducing neurons were occasionally found. No significant differences in hCDKL5 mRNA-positive neurons were observed in the motor cortex of the NHPs in this study.

[0215] Pathological examination revealed no gross lesions in the tissues or NHP. Those receiving the highest dose of NHP showed mild signs of inflammatory cell infiltration in the liver. Additionally, all NHP patients exhibited mild to moderate spinal cord axonal lesions and DRG satellite status.

[0216] Additionally, the expression and safety of NHP in the AAVrh91.UbC.CDKL5-1co.miR183 and AAVrh91.CBh.CDKL5-1co.miR183 vectors were examined. The expression was performed via the ICM pathway at a rate of 3 × 10¹⁴ oz. 10 The GC-based vector was administered to C. rhesus monkeys. Pathological analysis and neurological examinations were performed to assess the effect of AAV vector administration on CDKL5 expression.

[0217] In summary, the AAV-CDKL5 vector (SEQ ID NO:1) examined in this study can be used to achieve stable CDKL5 protein expression in neurons. AAV-CDKL5 gene therapy significantly improved the phenotype of the CDD mouse model. Furthermore, the AAV-CDKL5 vector can be efficiently delivered to non-human primates via the cerebellomedullary cistern and expressed throughout the CNS.

[0218] References

[0219] 1. Bahi-Buisson, N. et al. Key clinical features for identifying girls with CDKL5 mutations. Brain 131, 2647-2661, (2008).

[0220] 2. Fehr, S. et al. The CDKL5 disorder is an independent clinical entity associated with early-onset encephalopathy. European Journal of Human Genetics 21, 266-273, (2013).

[0221] 3. Kalscheuer, VM et al. Disruption of the serine / threonine kinase 9 gene causes severe X-linked infantile spasms and mental retardation. American Journal of Human Genetics 72, 1401-1411, (2003).

[0222] 4. Tao, J. et al. Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5 / STK9) gene are associated with severe neurodevelopmental retardation. American Journal of Human Genetics 75, 1149-1154, (2004).

[0223] 5. Weaving, LS et al., CDKL5 mutations cause a severe neurodevelopmental disorder with infantile spasms and mental retardation. American Journal of Human Genetics 75, 1079-1093, (2004).

[0224] 6. Characterization of human and mouse CDKL5 transcript isotypes by Hector, RD et al. PLOS ONE 11, e0157758, (2016).

[0225] 7. Baltussen, LL et al. (2018). "Chemical genetic identification of CDKL5 substrates reveals its role in neuronal microtubule dynamics". EMBO Journal 37(24).

[0226] 8. Munoz, I.M. et al. (2018). "Phosphoproteomic screening identifies physiological substrates of the CDKL5 kinase". Journal of the European Food Safety Authority.

[0227] All documents referenced in this specification are incorporated herein by reference, such as U.S. Provisional Patent Application No. 63 / 016,036, filed April 27, 2020; U.S. Provisional Patent Application No. 63 / 091,032, filed October 13, 2020; and U.S. Provisional Patent Application No. 63 / 109,608, filed November 4, 2020. The sequence list marked “20-9196PCT_SeqListing_ST25.txt” and the sequences and text therein, which are filed herein, are incorporated by reference. While the invention has been described with reference to specific embodiments, it should be understood that modifications may be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims. sequence list <110> University of Pennsylvania Trustees <120> Compositions that can be used to treat CDKL5 deficiency (CDD) <130> UPN-20-9196.PCT <150> US 63 / 016,036 <151> 2020-04-27 <150> US 63 / 091,032 <151> 2020-10-13 <150> US 63 / 109,608 <151> 2020-11-04 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 4634 <212> DNA <213> Artificial sequence <220> <223> Expression Box <220> <221> repeat_region <222> (1)..(130) <223> 5'ITR <220> <221> misc_feature <222> (130) (168) <223> AAV2 sequence upstream of 3' ITR <220> <221> promoter <222> (213)..(678) <223> Human synaptic protein <220> <221> CDS <222> (699)..(3581) <223> hCDKL5_1 <220> <221> misc_feature <222> (3596)...(4183) <223> WRPE <220> <221> polyA_signal <222> (4201)..(4439) <223> SV40 polyA <220> <221> misc_feature <222> (4467)...(44505) <223> AAV2 sequence upstream of 3' ITR <220> <221> repeat_region <222> (4505)...(4634) <223> 3'ITR <400> 1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggaagatcc tctagaacta tagctagcat gcctgcagag ggccctgcgt atgagtgcaa 240 gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac 300 ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg 360 aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg 420 ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg 480 tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc 540 gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat 600 ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc 660 gtgtcgtgcc tgagagcgca gtcgaattcg ccgccacc atg aag atc ccc aac atc 716 ggc aac gtg atg aac aag ttc gag atc ctg ggc gtt gtc gga gaa ggc 764 gct tat ggc gtg gtg ctg aag tgc aga cac aaa gaa acc cac gag atc 812 gtg gcc atc aag aag ttc aag gac agc gaa gag aac gag gaa gtg aaa 860 gag aca acc ctg cgc gag ctg aag atg ctg aga acc ctg aag caa gag 908 aac atc gtg gaa ctg aaa gag gcc ttc cgg cgg aga ggc aag ctg tac 956 ctg gtg ttc gag tac gtg gaa aag aac andg ctc gag ctg ctg gaa gag 1004 atg ccc aat ggc gtg cca cct gag aaa gtg aag tcc tac atc tac cag 1052 ctg atc aag gcc atc cac tgg tgc cac aag aac gac atc gtg cac aga 1100 gac atc aag ccc gag aac ctg ctg atc tcc cac aac gac gtg ctg aag 1148 ctg tgc gac ttc ggc ttc gcc aga aat ctg agc gag ggc aac aac gcc 1196 aac tac acc gag tat gtg gcc acc aga tgg tac aga agc cct gaa ctg 1244 ctg ctg gga gcc cct tac ggc aag tct gtg gat atg tgg tcc gtg ggc 1292 tgt atc ctg gga gag ctg tct gat ggc caa cct ctg ttt cct ggc gag 1340 tcc gag atc gac cag ctg ttc aca atc ​​cag aag gtg ctg ggc cct ctg 1388 cct agc gag cag atg aag ctg ttc tac agc aac ccc aga ttc cac ggc 1436 ctg aga ttc cct gcc gtg aat cac cct cag agc ctg gaa aga aga tac 1484 ctg ggc atc ctg aac agc gtg ctg ctg gac ctg atg aag aat ctg ctg 1532 aaa ctg gac ccc gcc gac aga tac ctg acc gag cag tgt ctg aac cat 1580 cct acc ttt cag acc cag aga ctg ctg gat aga agc ccc agc aga agc 1628 gcc aag aga aag ccc tac cac gtg gaa agc agc acc ctg agc aat aga 1676 aac cag gcc gga aag agc act gcc ctg cag agc cac cac aga agc aac 1724 agc aag gac atc cag aac ctg agc gtg ggc ctg cct aga gct gat gaa 1772 gga ctg cct gcc aac gag agc ttc ctg aat gga aat ctg gct ggc gcc 1820 tct ctg agc cct ctg cac aca aag aca tac cag gcc agc agc cag cct 1868 ggc agc aca agc aag gac ctg acc aac aac aac atc cct cat ctg ctg 1916 agc ccc aaa gag gcc aag agc aag acc gag ttc gac ttc aac atc gac 1964 ccc aag cct agc gaa ggc cct ggc acc aag tac ctg aag tcc aac tcc 2012 aga agc cag cag aac cgg cac tcc ttc atg gaa agc tct cag agc aag 2060 gcc ggc aca ctg cag cct aac gag aag cag agc aga cac agc tac atc 2108 gac aca atc cct cag tcc tct cgg agc ccc agc tac aga aca aag gcc 2156 aaa tct cac ggc gcc ctg agc gat agc aag agc gtg tcc aat ctg tcc 2204 gag gct aga gcc cag atc gcc gag cct agc acc agc aga tac ttc cct 2252 agc agc tgc ctg gat ctg aac agc cct aca agc ccc aca cct acc agg 2300 cac agc gat acc aga aca ctg ctg tcc cca agc ggc cgg aac aac aga 2348 aat gag ggc acc ctg gac agc aga cgg acc acc aca aga cac agc aag 2396 acc atg gaa gaa ctg aag ctg ccc gag cac atg gac tct agc cac tct 2444 cat tct ctg agc gcc cct cac gag tcc ttt agc tac ggc ctg ggc tac 2492 aca agc cca ttt tcc agt cag cag cgg ccc cac aga cac tct atg tac 2540 gtg acc aga gac aaa gtc cgc gcc aaa ggc ctg gat ggc tct ctg tct 2588 att gga caa ggc atg gcc gcc aga gcc aat agc ctg caa ctt ctg tct 2636 cct cag cct ggc gaa cag ctg cct cct gaa atg aca gtg gcc cgg tcc 2684 agc gtg aaa gaa acc agc aga gag ggc acc tcc agc ttc cac acc aga 2732 cag aaa agc gaa ggc ggc gtg tac cac gat cct cac tct gat gat ggc 2780 aca gcc cct aaa gag aac aga cac ctg tac aac gac ccc gtg cct aga 2828 aga gtg ggc tcc ttc tac aga gtg cct tct cct aga cct gac aac tcc 2876 ttc cac gag aac aac gtg tca acc cgg gtg tcc agc ctg cct tct gag 2924 agc agc tct ggc aca aac cac tcc aag agg cag ccc gcc ttc gat cct 2972 tgg aag tcc cct gag aat atc agc cac tcc gag cag ctg aaa gag aaa 3020 gag aag cag ggc ttt ttc cgg tcc atg aag aag aag aaa aag aag tcc 3068 cag aca gtc ccc aac agc gac agc cct gat ctc ctg aca ctg cag aag 3116 tcc atc cac agc gcc agc aca cct agc agc aga cct aaa gag tgg cgg 3164 cca gag aag atc agc gac ctg caa aca cag agc cag cct ctg aag tcc 3212 ctg agg aag ctg ctg cat ctg tcc tcc gcc tct aat cac cct gcc agc 3260 agc gat ccc aga ttt cag cca ctg aca gcc cag cag acc aag aac tcc 3308 ttc agc gag atc cgg att cac cca ctg tct cag gcc agc ggc ggc agc 3356 tct aat atc aga caa gag ccc gct cct aag ggc aga cct gct ctc caa 3404 ctg cct gga cag atg gat cct ggc tgg cac gtg tcc tct gtg aca aga 3452 tct gcc acc gag gga cct agc tat tct gag cag ctg ggc gcc aag tct 3500 ggc ccc aat gga cac ccc tac aac cgg acc aat cgg agc aga atg ccc 3548 aac ctg aac gac ctc aaa gaa aca gcc ctc tga gagcttatcg ataatcaacc 3601 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 3661 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 3721 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 3781 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3841 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3901 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3961 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 4021 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 4081 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgcttcg 4141 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgatcgag 4201 gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat 4261 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 4321 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca 4381 gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga 4441 taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact 4501 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg 4561 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg 4621 agcgagcgcg cag 4634 <210> 2 <211> 960 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 2 Met Lys Ile Pro Asn Ile Gly Asn Val Met Asn Lys Phe Glu Ile Leu 1 5 10 15 Gly Val Val Gly Glu Gly Ala Tyr Gly Val Val Leu Lys Cys Arg His 20 25 30 Lys Glu Thr His Glu Ile Val Ala Ile Lys Lys Phe Lys Asp Ser Glu 35 40 45 Glu Asn Glu Glu Val Lys Glu Thr Thr Leu Arg Glu Leu Lys Met Leu 50 55 60 Arg Thr Leu Lys Gln Glu Asn Ile Val Glu Leu Lys Glu Ala Phe Arg 65 70 75 80 Arg Arg Gly Lys Leu Tyr Leu Val Phe Glu Tyr Val Glu Lys Asn Met 85 90 95 Leu Glu Leu Leu Glu Glu Met Pro Asn Gly Val Pro Pro Glu Lys Val 100 105 110 Lys Ser Tyr Ile Tyr Gln Leu Ile Lys Ala Ile His Trp Cys His Lys 115 120 125 Asn Asp Ile Val His Arg Asp Ile Lys Pro Glu Asn Leu Leu Ile Ser 130 135 140 His Asn Asp Val Leu Lys Leu Cys Asp Phe Gly Phe Ala Arg Asn Leu 145 150 155 160 Ser Glu Gly Asn Asn Ala Asn Tyr Thr Glu Tyr Val Ala Thr Arg Trp 165 170 175 Tyr Arg Ser Pro Glu Leu Leu Leu Gly Ala Pro Tyr Gly Lys Ser Val 180 185 190 Asp Met Trp Ser Val Gly Cys Ile Leu Gly Glu Leu Ser Asp Gly Gln 195 200 205 Pro Leu Phe Pro Gly Glu Ser Glu Ile Asp Gln Leu Phe Thr Ile Gln 210 215 220 Lys Val Leu Gly Pro Leu Pro Ser Glu Gln Met Lys Leu Phe Tyr Ser 225 230 235 240 Asn Pro Arg Phe His Gly Leu Arg Phe Pro Ala Val Asn His Pro Gln 245 250 255 Ser Leu Glu Arg Arg Tyr Leu Gly Ile Leu Asn Ser Val Leu Leu Asp 260 265 270 Leu Met Lys Asn Leu Leu Lys Leu Asp Pro Ala Asp Arg Tyr Leu Thr 275 280 285 Glu Gln Cys Leu Asn His Pro Thr Phe Gln Thr Gln Arg Leu Leu Asp 290 295 300 Arg Ser Pro Ser Arg Ser Ala Lys Arg Lys Pro Tyr His Val Glu Ser 305 310 315 320 Ser Thr Leu Ser Asn Arg Asn Gln Ala Gly Lys Ser Thr Ala Leu Gln 325 330 335 Ser His His Arg Ser Asn Ser Lys Asp Ile Gln Asn Leu Ser Val Gly 340 345 350 Leu Pro Arg Ala Asp Glu Gly Leu Pro Ala Asn Glu Ser Phe Leu Asn 355 360 365 Gly Asn Leu Ala Gly Ala Ser Leu Ser Pro Leu His Thr Lys Thr Tyr 370 375 380 Gln Ala Ser Ser Gln Pro Gly Ser Thr Ser Lys Asp Leu Thr Asn Asn 385 390 395 400 Asn Ile Pro His Leu Leu Ser Pro Lys Glu Ala Lys Ser Lys Thr Glu 405 410 415 Phe Asp Phe Asn Ile Asp Pro Lys Pro Ser Glu Gly Pro Gly Thr Lys 420 425 430 Tyr Leu Lys Ser Asn Ser Arg Ser Gln Gln Asn Arg His Ser Phe Met 435 440 445 Glu Ser Ser Gln Ser Lys Ala Gly Thr Leu Gln Pro Asn Glu Lys Gln 450 455 460 Ser Arg His Ser Tyr Ile Asp Thr Ile Pro Gln Ser Ser Arg Ser Pro 465 470 475 480 Ser Tyr Arg Thr Lys Ala Lys Ser His Gly Ala Leu Ser Asp Ser Lys 485 490 495 Ser Val Ser Asn Leu Ser Glu Ala Arg Ala Gln Ile Ala Glu Pro Ser 500 505 510 Thr Ser Arg Tyr Phe Pro Ser Ser Cys Leu Asp Leu Asn Ser Pro Thr 515 520 525 Ser Pro Thr Pro Thr Arg His Ser Asp Thr Arg Thr Leu Leu Ser Pro 530 535 540 Ser Gly Arg Asn Asn Arg Asn Glu Gly Thr Leu Asp Ser Arg Arg Thr 545 550 555 560 Thr Thr Arg His Ser Lys Thr Met Glu Glu Leu Lys Leu Pro Glu His 565 570 575 Met Asp Ser Ser His Ser His Ser Leu Ser Ala Pro His Glu Ser Phe 580 585 590 Ser Tyr Gly Leu Gly Tyr Thr Ser Pro Phe Ser Ser Gln Gln Arg Pro 595 600 605 His Arg His Ser Met Tyr Val Thr Arg Asp Lys Val Arg Ala Lys Gly 610 615 620 Leu Asp Gly Ser Leu Ser Ile Gly Gln Gly Met Ala Ala Arg Ala Asn 625 630 635 640 Ser Leu Gln Leu Leu Ser Pro Gln Pro Gly Glu Gln Leu Pro Pro Glu 645 650 655 Met Thr Val Ala Arg Ser Ser Val Lys Glu Thr Ser Arg Glu Gly Thr 660 665 670 Ser Ser Phe His Thr Arg Gln Lys Ser Glu Gly Gly Val Tyr His Asp 675 680 685 Pro His Ser Asp Asp Gly Thr Ala Pro Lys Glu Asn Arg His Leu Tyr 690 695 700 Asn Asp Pro Val Pro Arg Arg Val Gly Ser Phe Tyr Arg Val Pro Ser 705 710 715 720 Pro Arg Pro Asp Asn Ser Phe His Glu Asn Asn Val Ser Thr Arg Val 725 730 735 Ser Ser Leu Pro Ser Glu Ser Ser Ser Gly Thr Asn His Ser Lys Arg 740 745 750 Gln Pro Ala Phe Asp Pro Trp Lys Ser Pro Glu Asn Ile Ser His Ser 755 760 765 Glu Gln Leu Lys Glu Lys Glu Lys Gln Gly Phe Phe Arg Ser Met Lys 770 775 780 Light Light Light Light Light Ser Gln Thr Val Pro Asn Ser Asp Ser Pro Asp 785 790 795 800 Leu Leu Thr Leu Gln Lys Ser Ile His Ser Ala Ser Thr Pro Ser Ser 805 810 815 Arg Pro Lys Glu Trp Arg Pro Glu Lys Ile Ser Asp Leu Gln Thr Gln 820 825 830 Ser Gln Pro Leu Lys Ser Leu Arg Lys Leu Leu His Leu Ser Ser Ala 835 840 845 Ser Asn His Pro Ala Ser Ser Asp Pro Arg Phe Gln Pro Leu Thr Ala 850 855 860 Gln Gln Thr Lys Asn Ser Phe Ser Glu Ile Arg Ile His Pro Leu Ser 865 870 875 880 Gln Ala Ser Gly Gly Ser Ser Asn Ile Arg Gln Glu Pro Ala Pro Lys 885 890 895 Gly Arg Pro Ala Leu Gln Leu Pro Gly Gln Met Asp Pro Gly Trp His 900 905 910 Val Ser Ser Val Thr Arg Ser Ala Thr Glu Gly Pro Ser Tyr Ser Glu 915 920 925 Gln Leu Gly Ala Lys Ser Gly Pro Asn Gly His Pro Tyr Asn Arg Thr 930 935 940 Asn Arg Ser Arg Met Pro Asn Leu Asn Asp Leu Lys Glu Thr Ala Leu 945 950 955 960 <210> 3 <211> 4750 <212> DNA <213> Artificial sequence <220> <223> Expression cassette <220> <221> repeat_region <222> (1)..(130) <223> 5'ITR <220> <221> misc_feature <222> (130) (168) <223> AAV2 sequence upstream of 3' ITR <220> <221> promoter <222> (213)..(678) <223> Human synaptic protein <220> <221> CDS <222> (699)..(3581) <223> hCDKL5-1 <220> <221> misc_binding <222> (3590)...(3705) <223> miR183 <220> <221> misc_feature <222> (3712)..(4299) <223> WRPE <220> <221> polyA_signal <222> (4317)...(4555) <223> SV40 polyA <220> <221> misc_feature <222> (4583)...(4621) <223> AAV2 sequence upstream of 3' ITR <220> <221> repeat_region <222> (4621)...(4750) <223> 3'ITR <400> 3 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggaagatcc tctagaacta tagctagcat gcctgcagag ggccctgcgt atgagtgcaa 240 gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac 300 ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg 360 aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg 420 ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg 480 tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc 540 gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat 600 ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc 660 gtgtcgtgcc tgagagcgca gtcgaattcg ccgccacc atg aag atc ccc aac atc 716 ggc aac gtg atg aac aag ttc gag atc ctg ggc gtt gtc gga gaa ggc 764 gct tat ggc gtg gtg ctg aag tgc aga cac aaa gaa acc cac gag atc 812 gtg gcc atc aag aag ttc aag gac agc gaa gag aac gag gaa gtg aaa 860 gag aca acc ctg cgc gag ctg aag atg ctg aga acc ctg aag caa gag 908 aac atc gtg gaa ctg aaa gag gcc ttc cgg cgg aga ggc aag ctg tac 956 ctg gtg ttc gag tac gtg gaa aag aac andg ctc gag ctg ctg gaa gag 1004 atg ccc aat ggc gtg cca cct gag aaa gtg aag tcc tac atc tac cag 1052 ctg atc aag gcc atc cac tgg tgc cac aag aac gac atc gtg cac aga 1100 gac atc aag ccc gag aac ctg ctg atc tcc cac aac gac gtg ctg aag 1148 ctg tgc gac ttc ggc ttc gcc aga aat ctg agc gag ggc aac aac gcc 1196 aac tac acc gag tat gtg gcc acc aga tgg tac aga agc cct gaa ctg 1244 ctg ctg gga gcc cct tac ggc aag tct gtg gat atg tgg tcc gtg ggc 1292 tgt atc ctg gga gag ctg tct gat ggc caa cct ctg ttt cct ggc gag 1340 tcc gag atc gac cag ctg ttc aca atc ​​cag aag gtg ctg ggc cct ctg 1388 cct agc gag cag atg aag ctg ttc tac agc aac ccc aga ttc cac ggc 1436 ctg aga ttc cct gcc gtg aat cac cct cag agc ctg gaa aga aga tac 1484 ctg ggc atc ctg aac agc gtg ctg ctg gac ctg atg aag aat ctg ctg 1532 aaa ctg gac ccc gcc gac aga tac ctg acc gag cag tgt ctg aac cat 1580 cct acc ttt cag acc cag aga ctg ctg gat aga agc ccc agc aga agc 1628 gcc aag aga aag ccc tac cac gtg gaa agc agc acc ctg agc aat aga 1676 aac cag gcc gga aag agc act gcc ctg cag agc cac cac aga agc aac 1724 agc aag gac atc cag aac ctg agc gtg ggc ctg cct aga gct gat gaa 1772 gga ctg cct gcc aac gag agc ttc ctg aat gga aat ctg gct ggc gcc 1820 tct ctg agc cct ctg cac aca aag aca tac cag gcc agc agc cag cct 1868 ggc agc aca agc aag gac ctg acc aac aac aac atc cct cat ctg ctg 1916 agc ccc aaa gag gcc aag agc aag acc gag ttc gac ttc aac atc gac 1964 ccc aag cct agc gaa ggc cct ggc acc aag tac ctg aag tcc aac tcc 2012 aga agc cag cag aac cgg cac tcc ttc atg gaa agc tct cag agc aag 2060 gcc ggc aca ctg cag cct aac gag aag cag agc aga cac agc tac atc 2108 gac aca atc cct cag tcc tct cgg agc ccc agc tac aga aca aag gcc 2156 aaa tct cac ggc gcc ctg agc gat agc aag agc gtg tcc aat ctg tcc 2204 gag gct aga gcc cag atc gcc gag cct agc acc agc aga tac ttc cct 2252 agc agc tgc ctg gat ctg aac agc cct aca agc ccc aca cct acc agg 2300 cac agc gat acc aga aca ctg ctg tcc cca agc ggc cgg aac aac aga 2348 aat gag ggc acc ctg gac agc aga cgg acc acc aca aga cac agc aag 2396 acc atg gaa gaa ctg aag ctg ccc gag cac atg gac tct agc cac tct 2444 cat tct ctg agc gcc cct cac gag tcc ttt agc tac ggc ctg ggc tac 2492 aca agc cca ttt tcc agt cag cag cgg ccc cac aga cac tct atg tac 2540 gtg acc aga gac aaa gtc cgc gcc aaa ggc ctg gat ggc tct ctg tct 2588 att gga caa ggc atg gcc gcc aga gcc aat agc ctg caa ctt ctg tct 2636 cct cag cct ggc gaa cag ctg cct cct gaa atg aca gtg gcc cgg tcc 2684 agc gtg aaa gaa acc agc aga gag ggc acc tcc agc ttc cac acc aga 2732 cag aaa agc gaa ggc ggc gtg tac cac gat cct cac tct gat gat ggc 2780 aca gcc cct aaa gag aac aga cac ctg tac aac gac ccc gtg cct aga 2828 aga gtg ggc tcc ttc tac aga gtg cct tct cct aga cct gac aac tcc 2876 ttc cac gag aac aac gtg tca acc cgg gtg tcc agc ctg cct tct gag 2924 agc agc tct ggc aca aac cac tcc aag agg cag ccc gcc ttc gat cct 2972 tgg aag tcc cct gag aat atc agc cac tcc gag cag ctg aaa gag aaa 3020 gag aag cag ggc ttt ttc cgg tcc atg aag aag aag aaa aag aag tcc 3068 cag aca gtc ccc aac agc gac agc cct gat ctc ctg aca ctg cag aag 3116 tcc atc cac agc gcc agc aca cct agc agc aga cct aaa gag tgg cgg 3164 cca gag aag atc agc gac ctg caa aca cag agc cag cct ctg aag tcc 3212 ctg agg aag ctg ctg cat ctg tcc tcc gcc tct aat cac cct gcc agc 3260 agc gat ccc aga ttt cag cca ctg aca gcc cag cag acc aag aac tcc 3308 ttc agc gag atc cgg att cac cca ctg tct cag gcc agc ggc ggc agc 3356 tct aat atc aga caa gag ccc gct cct aag ggc aga cct gct ctc caa 3404 ctg cct gga cag atg gat cct ggc tgg cac gtg tcc tct gtg aca aga 3452 tct gcc acc gag gga cct agc tat tct gag cag ctg ggc gcc aag tct 3500 ggc ccc aat gga cac ccc tac aac cgg acc aat cgg agc aga atg ccc 3548 aac ctg aac gac ctc aaa gaa aca gcc ctc tga gagcttatac cggtagtgaa 3601 ttctaccagt gccataggat agtgaattct accagtgcca tacacgtgag tgaattctac 3661 cagtgccata gcatgcagtg aattctacca gtgccatagg tacccgataa tcaacctctg 3721 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta 3781 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt 3841 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gccgttgtc 3901 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt 3961 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccct tgccacggcg 4021 gaactcatcg ccgcctgcct tgccgctgc tggacagggg ctcggctgtt gggcactgac 4081 aattccgtgg tgttgtcggg gaaatcatcg tctttcctt ggctgctcgc ctgtgttgcc 4141 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac 4201 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg cttcgccct 4261 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg atcgaggccg 4321 cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag 4381 tgaaaaaaat gctttattg tgaaatttgt gatgctattg ctttatttgt aaccattata 4441 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg 4501 gagatgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag 4561 gatcttccta gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa 4621 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc 4681 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 4741 agcgcgcag 4750 <210> 4 <211> 960 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 4 Met Lys Ile Pro Asn Ile Gly Asn Val Met Asn Lys Phe Glu Ile Leu 1 5 10 15 Gly Val Val Gly Glu Gly Ala Tyr Gly Val Val Leu Lys Cys Arg His 20 25 30 Lys Glu Thr His Glu Ile Val Ala Ile Lys Lys Phe Lys Asp Ser Glu 35 40 45 Glu Asn Glu Glu Val Lys Glu Thr Thr Leu Arg Glu Leu Lys Met Leu 50 55 60 Arg Thr Leu Lys Gln Glu Asn Ile Val Glu Leu Lys Glu Ala Phe Arg 65 70 75 80 Arg Arg Gly Lys Leu Tyr Leu Val Phe Glu Tyr Val Glu Lys Asn Met 85 90 95 Leu Glu Leu Leu Glu Glu Met Pro Asn Gly Val Pro Pro Glu Lys Val 100 105 110 Lys Ser Tyr Ile Tyr Gln Leu Ile Lys Ala Ile His Trp Cys His Lys 115 120 125 Asn Asp Ile Val His Arg Asp Ile Lys Pro Glu Asn Leu Leu Ile Ser 130 135 140 His Asn Asp Val Leu Lys Leu Cys Asp Phe Gly Phe Ala Arg Asn Leu 145 150 155 160 Ser Glu Gly Asn Asn Ala Asn Tyr Thr Glu Tyr Val Ala Thr Arg Trp 165 170 175 Tyr Arg Ser Pro Glu Leu Leu Leu Gly Ala Pro Tyr Gly Lys Ser Val 180 185 190 Asp Met Trp Ser Val Gly Cys Ile Leu Gly Glu Leu Ser Asp Gly Gln 195 200 205 Pro Leu Phe Pro Gly Glu Ser Glu Ile Asp Gln Leu Phe Thr Ile Gln 210 215 220 Lys Val Leu Gly Pro Leu Pro Ser Glu Gln Met Lys Leu Phe Tyr Ser 225 230 235 240 Asn Pro Arg Phe His Gly Leu Arg Phe Pro Ala Val Asn His Pro Gln 245 250 255 Ser Leu Glu Arg Arg Tyr Leu Gly Ile Leu Asn Ser Val Leu Leu Asp 260 265 270 Leu Met Lys Asn Leu Leu Lys Leu Asp Pro Ala Asp Arg Tyr Leu Thr 275 280 285 Glu Gln Cys Leu Asn His Pro Thr Phe Gln Thr Gln Arg Leu Leu Asp 290 295 300 Arg Ser Pro Ser Arg Ser Ala Lys Arg Lys Pro Tyr His Val Glu Ser 305 310 315 320 Ser Thr Leu Ser Asn Arg Asn Gln Ala Gly Lys Ser Thr Ala Leu Gln 325 330 335 Ser His His Arg Ser Asn Ser Lys Asp Ile Gln Asn Leu Ser Val Gly 340 345 350 Leu Pro Arg Ala Asp Glu Gly Leu Pro Ala Asn Glu Ser Phe Leu Asn 355 360 365 Gly Asn Leu Ala Gly Ala Ser Leu Ser Pro Leu His Thr Lys Thr Tyr 370 375 380 Gln Ala Ser Ser Gln Pro Gly Ser Thr Ser Lys Asp Leu Thr Asn Asn 385 390 395 400 Asn Ile Pro His Leu Leu Ser Pro Lys Glu Ala Lys Ser Lys Thr Glu 405 410 415 Phe Asp Phe Asn Ile Asp Pro Lys Pro Ser Glu Gly Pro Gly Thr Lys 420 425 430 Tyr Leu Lys Ser Asn Ser Arg Ser Gln Gln Asn Arg His Ser Phe Met 435 440 445 Glu Ser Ser Gln Ser Lys Ala Gly Thr Leu Gln Pro Asn Glu Lys Gln 450 455 460 Ser Arg His Ser Tyr Ile Asp Thr Ile Pro Gln Ser Ser Arg Ser Pro 465 470 475 480 Ser Tyr Arg Thr Lys Ala Lys Ser His Gly Ala Leu Ser Asp Ser Lys 485 490 495 Ser Val Ser Asn Leu Ser Glu Ala Arg Ala Gln Ile Ala Glu Pro Ser 500 505 510 Thr Ser Arg Tyr Phe Pro Ser Ser Cys Leu Asp Leu Asn Ser Pro Thr 515 520 525 Ser Pro Thr Pro Thr Arg His Ser Asp Thr Arg Thr Leu Leu Ser Pro 530 535 540 Ser Gly Arg Asn Asn Arg Asn Glu Gly Thr Leu Asp Ser Arg Arg Thr 545 550 555 560 Thr Thr Arg His Ser Lys Thr Met Glu Glu Leu Lys Leu Pro Glu His 565 570 575 Met Asp Ser Ser His Ser His Ser Leu Ser Ala Pro His Glu Ser Phe 580 585 590 Ser Tyr Gly Leu Gly Tyr Thr Ser Pro Phe Ser Ser Gln Gln Arg Pro 595 600 605 His Arg His Ser Met Tyr Val Thr Arg Asp Lys Val Arg Ala Lys Gly 610 615 620 Leu Asp Gly Ser Leu Ser Ile Gly Gln Gly Met Ala Ala Arg Ala Asn 625 630 635 640 Ser Leu Gln Leu Leu Ser Pro Gln Pro Gly Glu Gln Leu Pro Pro Glu 645 650 655 Met Thr Val Ala Arg Ser Ser Val Lys Glu Thr Ser Arg Glu Gly Thr 660 665 670 Ser Ser Phe His Thr Arg Gln Lys Ser Glu Gly Gly Val Tyr His Asp 675 680 685 Pro His Ser Asp Asp Gly Thr Ala Pro Lys Glu Asn Arg His Leu Tyr 690 695 700 Asn Asp Pro Val Pro Arg Arg Val Gly Ser Phe Tyr Arg Val Pro Ser 705 710 715 720 Pro Arg Pro Asp Asn Ser Phe His Glu Asn Asn Val Ser Thr Arg Val 725 730 735 Ser Ser Leu Pro Ser Glu Ser Ser Ser Gly Thr Asn His Ser Lys Arg 740 745 750 Gln Pro Ala Phe Asp Pro Trp Lys Ser Pro Glu Asn Ile Ser His Ser 755 760 765 Glu Gln Leu Lys Glu Lys Glu Lys Gln Gly Phe Phe Arg Ser Met Lys 770 775 780 Light Light Light Light Light Ser Gln Thr Val Pro Asn Ser Asp Ser Pro Asp 785 790 795 800 Leu Leu Thr Leu Gln Lys Ser Ile His Ser Ala Ser Thr Pro Ser Ser 805 810 815 Arg Pro Lys Glu Trp Arg Pro Glu Lys Ile Ser Asp Leu Gln Thr Gln 820 825 830 Ser Gln Pro Leu Lys Ser Leu Arg Lys Leu Leu His Leu Ser Ser Ala 835 840 845 Ser Asn His Pro Ala Ser Ser Asp Pro Arg Phe Gln Pro Leu Thr Ala 850 855 860 Gln Gln Thr Lys Asn Ser Phe Ser Glu Ile Arg Ile His Pro Leu Ser 865 870 875 880 Gln Ala Ser Gly Gly Ser Ser Asn Ile Arg Gln Glu Pro Ala Pro Lys 885 890 895 Gly Arg Pro Ala Leu Gln Leu Pro Gly Gln Met Asp Pro Gly Trp His 900 905 910 Val Ser Ser Val Thr Arg Ser Ala Thr Glu Gly Pro Ser Tyr Ser Glu 915 920 925 Gln Leu Gly Ala Lys Ser Gly Pro Asn Gly His Pro Tyr Asn Arg Thr 930 935 940 Asn Arg Ser Arg Met Pro Asn Leu Asn Asp Leu Lys Glu Thr Ala Leu 945 950 955 960 <210> 5 <211> 4757 <212> DNA <213> Artificial sequence <220> <223> Expression cassette <220> <221> repeat_region <222> (1)..(130) <223> 5'ITR <220> <221> misc_feature <222> (130) (168) <223> AAV2 sequence upstream of 3' ITR <220> <221> promoter <222> (213)..(678) <223> Human synaptic protein <220> <221> misc_feature <222> (690) (695) <223> Kozak <220> <221> CDS <222> (696) (3704) <223> hCDKL5-2GS <220> <221> misc_feature <222> (3719)...(4306) <223> WRPE <220> <221> polyA_signal <222> (4324)...(4562) <223> SV40 polyA <220> <221> misc_feature <222> (4590)...(4628) <223> AAV2 sequence upstream of 3' ITR <220> <221> repeat_region <222> (4628)...(4757) <223> 3'ITR <400> 5 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggaagatcc tctagaacta tagctagcat gcctgcagag ggccctgcgt atgagtgcaa 240 gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac 300 ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg 360 aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg 420 ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg 480 tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc 540 gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat 600 ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc 660 gtgtcgtgcc tgagagcgca gtcgaattcg ccacc atg aag atc ccc aat atc 713 ggc aac gtg atg aat aag ttc gag atc ctg gga gtg gtg gga gag gga 761 gca tac ggc gtg gtg ctg aag tgc aga cac aag gag aca cac gag atc 809 gtg gcc atc aag aag ttt aag gat agc gag gag aat gag gag gtg aag 857 gag aca acc ctg cgc gag ctg aag andg ctg cgg aca ctg aag cag gag 905 aac atc gtg gag ctg aag gag gcc ttc cgg aga agg ggc aag ctg tac 953 ctg gtg ttt gag tac gtg gag aag aac andg ctg gag ctg ctg gag gag 1001 atg cct aat ggc gtg ccc cct gag aag gtg aag agc tac atc tat cag 1049 ctg atc aag gcc atc cac tgg tgc cac aag aac gac atc gtg cac cgc 1097 gat atc aag cca gag aac ctg ctg atc agc cac aat gac gtg ctg aag 1145 ctg tgc gat ttc ggc ttt gcc cgg aac ctg tcc gag ggc aac aac gcc 1193 aat tac aca gag tac gtg gca acc agg tgg tac cgg tcc cct gag ctg 1241 ctg ctg gga gca cca tac ggc aag tct gtg gac atg tgg agc gtg ggc 1289 tgc atc ctg gga gag ctg tcc gac gga cag cca ctg ttc cct ggc gag 1337 tct gag atc gat cag ctg ttt acc atc cag aag gtg ctg ggc ccc ctg 1385 cct tcc gag cag atg aag ctg ttc tac tct aac cca agg ttc cac ggc 1433 ctg agg ttt cca gcc gtg aat cac ccc cag tct ctg gag cgc cgg tat 1481 ctg ggc atc ctg aac agc gtg ctg ctg gat ctg atg aag aat ctg ctg 1529 aag ctg gac cct gcc gat cgc tac ctg acc gag cag tgt ctg aat cac 1577 cca aca ttt cag acc cag aga ctg ctg gac agg agc ccc tcc cgc tct 1625 gcc aag agg aag cct tat cac gtg gag agc tcc acc ctg agc aat cgc 1673 aac cag gcc ggc aag tcc aca gcc ctg cag tct cac cac cgg agc aac 1721 tcc aag gac atc cag aat ctg agc gtg ggc ctg cca agg gca gac gag 1769 ggc ctg cct gcc aac gag tcc ttc ctg aat gga aac ctg gca gga gcc 1817 tct ctg agc cca ctg cac aca aag acc tac cag gcc tct agc cag cct 1865 ggc tcc aca tct aag gac ctg acc aac aat aac atc cct cac ctg ctg 1913 agc cca aag gag gcc aag tcc aag acc gag ttc gac ttt aat atc gat 1961 cca aag ccc agc gag ggc cca ggc aca aag tat ctg aag agc aac tcc 2009 aga tct cag cag aat agg cac tct ttc atg gag tcc tct cag agc aag 2057 gcc ggc acc ctg cag ccc aac gag aag cag agc agg cac tcc tac atc 2105 gat acc atc cca cag agc tcc cgc agc ccc tcc tat cgg aca aag gcc 2153 aag agc cac ggc gcc ctg tct gac agc aag tcc gtg tct aat ctg tcc 2201 gag gca aga gca cag atc gca gag cca agc acc tcc agg tac ttt ccc 2249 tct tcc tgt ctg gac ctg aac tct cca aca agc cct aca cca acc aga 2297 cac agc gat aca agg acc ctg ctg tct ccc agc ggc aga aat aac agg 2345 aac gag ggc acc ctg gat agc aga agg acc aca acc aga cac tcc aag 2393 aca atg gag gag ctg aag ctg ccc gag cac atg gac tcc tct cac tcc 2441 cac tct ctg agc gcc cct cac gag tcc ttc tct tac ggc ctg ggc tat 2489 acc agc cct ttt agc tcc cag cag cgc cca cac agg cac tcc atg tac 2537 gtg aca aga gac aag gtg agg gcc aag ggc ctg gac ggc agc ctg tcc 2585 atc gga cag gga atg gca gca agg gca aat agc ctg cag ctg ctg tcc 2633 cca cag cct gga gag cag ctg cca cca gag atg acc gtg gcc aga tct 2681 agc gtg aag gag aca agc agg gag ggc acc tcc tct ttc cac aca aga 2729 cag aag agc gag ggc ggc gtg tat cac gac cca cac tcc gac gac gga 2777 acc gca ccc aag gag aac agg cac ctg tac aac gat cca gtg ccc cgc 2825 cgg gtg ggc tct ttc tat cgg gtg cct agc cca cgg ccc gac aac tcc 2873 ttt cac gag aat aac gtg tct acc aga gtg agc tcc ctg cct agc gag 2921 tct agc tcc ggc aca aac cac tcc aag agg cag cca gcc ttt gat ccc 2969 tgg aag tct cct gag aat atc tct cac agc gag cag ctg aag gag aag 3017 gag aag cag ggc ttc ttt cgc agc atg aag aag aag aag aag aag agc 3065 cag aca acc gac agc acc aac ggc gag aat cct agc atc aag aag tcc 3113 ctg ttc cca ctg ttt aac tcc aag aat cac ctg aag cac tct agc tcc 3161 ctg aag aag ctg ccc gtg gtg aca cct cca atg gtg ccc aat tcc gac 3209 tct cct gat ctg ctg acc ctg cag aag agc atc cac agc gcc tcc aca 3257 cca tct agc aga ccc aag gag tgg agg cca gag aag atc tcc gat ctg 3305 cag acc cag tcc cag ccc ctg aag tct ctg cgg aag ctg ctg cac ctg 3353 tcc tct gcc agc aac cac cct gca agc tcc gac cct agg ttc cag cca 3401 ctg aca gcc cag cag acc aag aac tct ttt agc gag atc aga atc cac 3449 cca ctg tcc cag gcc tct gga ggc tct agc aac atc cgc cag gag cct 3497 gcc cca aag ggc cgg cca gcc ctg cag ctg cca gga cag atg gac ccc 3545 ggc tgg cac gtg tcc tct gtg aca aga tcc gcc acc gag gga cca tcc 3593 tac tct gag cag ctg gga gca aag tct gga cca aac gga cac cct tat 3641 aac agg acc aat aga tcc agg atg cct aat ctg aac gac ctg aag gag 3689 aca gcc ctg tga tag aagcttatcg ataatcaacc tctggattac aaaatttgtg 3744 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 3804 taatgcctt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 3864 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 3924 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 3984 tccttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 4044 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattc gtggtgttgt 4104 cggggaaatc atcgtcctttt cttggctgc tcgcctgtgt tgccacctgg attctgcgcg 4164 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tccgcggcc 4224 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct 4284 cccttgggc cgcctccccg catcgatacc gtcgatcgag gcgcttcga gcagacatga 4344 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 4404 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 4464 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt 4524 tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat 4584 ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg 4644 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc 4704 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cag 4757 <210> 6 <211> 1001 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 6 Met Lys Ile Pro Asn Ile Gly Asn Val Met Asn Lys Phe Glu Ile Leu 1 5 10 15 Gly Val Val Gly Glu Gly Ala Tyr Gly Val Val Leu Lys Cys Arg His 20 25 30 Lys Glu Thr His Glu Ile Val Ala Ile Lys Lys Phe Lys Asp Ser Glu 35 40 45 Glu Asn Glu Glu Val Lys Glu Thr Thr Leu Arg Glu Leu Lys Met Leu 50 55 60 Arg Thr Leu Lys Gln Glu Asn Ile Val Glu Leu Lys Glu Ala Phe Arg 65 70 75 80 Arg Arg Gly Lys Leu Tyr Leu Val Phe Glu Tyr Val Glu Lys Asn Met 85 90 95 Leu Glu Leu Leu Glu Glu Met Pro Asn Gly Val Pro Pro Glu Lys Val 100 105 110 Lys Ser Tyr Ile Tyr Gln Leu Ile Lys Ala Ile His Trp Cys His Lys 115 120 125 Asn Asp Ile Val His Arg Asp Ile Lys Pro Glu Asn Leu Leu Ile Ser 130 135 140 His Asn Asp Val Leu Lys Leu Cys Asp Phe Gly Phe Ala Arg Asn Leu 145 150 155 160 Ser Glu Gly Asn Asn Ala Asn Tyr Thr Glu Tyr Val Ala Thr Arg Trp 165 170 175 Tyr Arg Ser Pro Glu Leu Leu Leu Gly Ala Pro Tyr Gly Lys Ser Val 180 185 190 Asp Met Trp Ser Val Gly Cys Ile Leu Gly Glu Leu Ser Asp Gly Gln 195 200 205 Pro Leu Phe Pro Gly Glu Ser Glu Ile Asp Gln Leu Phe Thr Ile Gln 210 215 220 Lys Val Leu Gly Pro Leu Pro Ser Glu Gln Met Lys Leu Phe Tyr Ser 225 230 235 240 Asn Pro Arg Phe His Gly Leu Arg Phe Pro Ala Val Asn His Pro Gln 245 250 255 Ser Leu Glu Arg Arg Tyr Leu Gly Ile Leu Asn Ser Val Leu Leu Asp 260 265 270 Leu Met Lys Asn Leu Leu Lys Leu Asp Pro Ala Asp Arg Tyr Leu Thr 275 280 285 Glu Gln Cys Leu Asn His Pro Thr Phe Gln Thr Gln Arg Leu Leu Asp 290 295 300 Arg Ser Pro Ser Arg Ser Ala Lys Arg Lys Pro Tyr His Val Glu Ser 305 310 315 320 Ser Thr Leu Ser Asn Arg Asn Gln Ala Gly Lys Ser Thr Ala Leu Gln 325 330 335 Ser His His Arg Ser Asn Ser Lys Asp Ile Gln Asn Leu Ser Val Gly 340 345 350 Leu Pro Arg Ala Asp Glu Gly Leu Pro Ala Asn Glu Ser Phe Leu Asn 355 360 365 Gly Asn Leu Ala Gly Ala Ser Leu Ser Pro Leu His Thr Lys Thr Tyr 370 375 380 Gln Ala Ser Ser Gln Pro Gly Ser Thr Ser Lys Asp Leu Thr Asn Asn 385 390 395 400 Asn Ile Pro His Leu Leu Ser Pro Lys Glu Ala Lys Ser Lys Thr Glu 405 410 415 Phe Asp Phe Asn Ile Asp Pro Lys Pro Ser Glu Gly Pro Gly Thr Lys 420 425 430 Taurus Leu Lys Ser Asn Ser Arg Ser Gln Gln Asn Arg His Ser Phe Met 435 440 445 Glu Ser Ser Gln Ser Lys Ala Gly Thr Leu Gln Pro Asn Glu Lys Gln 450 455 460 Ser Arg His Ser Tyr Ile Asp Thr Ile Pro Gln Ser Ser Arg Ser Pro 465 470 475 480 Ser Tyr Arg Thr Lys Ala Lys Ser His Gly Ala Leu Ser Asp Ser Lys 485 490 495 Ser Val Ser Asn Leu Ser Glu Ala Arg Ala Gln Ile Ala Glu Pro Ser 500 505 510 Thr Ser Arg Tyr Phe Pro Ser Ser Cys Leu Asp Leu Asn Ser Pro Thr 515 520 525 Ser Pro Thr Pro Thr Arg His Ser Asp Thr Arg Thr Leu Leu Ser Pro 530 535 540 Ser Gly Arg Asn Asn Arg Asn Glu Gly Thr Leu Asp Ser Arg Arg Thr 545 550 555 560 Thr Thr Arg His Ser Lys Thr Met Glu Glu Leu Lys Leu Pro Glu His 565 570 575 Met Asp Ser Ser His Ser His Ser Leu Ser Ala Pro His Glu Ser Phe 580 585 590 Ser Tyr Gly Leu Gly Tyr Thr Ser Pro Phe Ser Ser Gln Gln Arg Pro 595 600 605 His Arg His Ser Met Tyr Val Thr Arg Asp Lys Val Arg Ala Lys Gly 610 615 620 Leu Asp Gly Ser Leu Ser Ile Gly Gln Gly Met Ala Ala Arg Ala Asn 625 630 635 640 Ser Leu Gln Leu Leu Ser Pro Gln Pro Gly Glu Gln Leu Pro Pro Glu 645 650 655 Met Thr Val Ala Arg Ser Ser Val Lys Glu Thr Ser Arg Glu Gly Thr 660 665 670 Ser Ser Phe His Thr Arg Gln Lys Ser Glu Gly Gly Val Tyr His Asp 675 680 685 Pro His Ser Asp Asp Gly Thr Ala Pro Lys Glu Asn Arg His Leu Tyr 690 695 700 Asn Asp Pro Val Pro Arg Arg Val Gly Ser Phe Tyr Arg Val Pro Ser 705 710 715 720 Pro Arg Pro Asp Asn Ser Phe His Glu Asn Asn Val Ser Thr Arg Val 725 730 735 Ser Ser Leu Pro Ser Glu Ser Ser Ser Gly Thr Asn His Ser Lys Arg 740 745 750 Gln Pro Ala Phe Asp Pro Trp Lys Ser Pro Glu Asn Ile Ser His Ser 755 760 765 Glu Gln Leu Lys Glu Lys Glu Lys Gln Gly Phe Phe Arg Ser Met Lys 770 775 780 Light Light Light Light Light Ser Gln Thr Thr Asp Ser Thr Asn Gly Glu Asn 785 790 795 800 Pro Ser Ile Lys Lys Ser Leu Phe Pro Leu Phe Asn Ser Lys Asn His 805 810 815 Leu Lys His Ser Ser Ser Leu Lys Lys Leu Pro Val Val Thr Pro Pro 820 825 830 Met Val Pro Asn Ser Asp Ser Pro Asp Leu Leu Thr Leu Gln Lys Ser 835 840 845 Ile His Ser Ala Ser Thr Pro Ser Ser Arg Pro Lys Glu Trp Arg Pro 850 855 860 Glu Lys Ile Ser Asp Leu Gln Thr Gln Ser Gln Pro Leu Lys Ser Leu 865 870 875 880 Arg Lys Leu Leu His Leu Ser Ser Ala Ser Asn His Pro Ala Ser Ser 885 890 895 Asp Pro Arg Phe Gln Pro Leu Thr Ala Gln Gln Thr Lys Asn Ser Phe 900 905 910 Ser Glu Ile Arg Ile His Pro Leu Ser Gln Ala Ser Gly Gly Ser Ser 915 920 925 Asn Ile Arg Gln Glu Pro Ala Pro Lys Gly Arg Pro Ala Leu Gln Leu 930 935 940 Pro Gly Gln Met Asp Pro Gly Trp His Val Ser Ser Val Thr Arg Ser 945 950 955 960 Ala Thr Glu Gly Pro Ser Tyr Ser Glu Gln Leu Gly Ala Lys Ser Gly 965 970 975 Pro Asn Gly His Pro Tyr Asn Arg Thr Asn Arg Ser Arg Met Pro Asn 980 985 990 Leu Asn Asp Leu Lys Glu Thr Ala Leu 995 1000 <210> 7 <211> 4583 <212> DNA <213> Artificial sequence <220> <223> Expression Box <220> <221> repeat_region <222> (1)..(130) <223> 5'ITR <220> <221> misc_feature <222> (130) (168) <223> AAV2 sequence upstream of 3' ITR <220> <221> promoter <222> (213)..(678) <223> Human synaptic protein <220> <221> misc_feature <222> (690) (695) <223> Kozak <220> <221> CDS <222> (696)..(3530) <223> hCDKL5-3GS <220> <221> misc_feature <222> (3545)..(4132) <223> WRPE <220> <221> polyA_signal <222> (4150)...(4388) <223> SV40 polyA <220> <221> misc_feature <222> (4416)...(4454) <223> AAV2 sequence upstream of 3' ITR <220> <221> repeat_region <222> (4454)..(4583) <223> 3'ITR <400> 7 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggaagatcc tctagaacta tagctagcat gcctgcagag ggccctgcgt atgagtgcaa 240 gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac 300 ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg 360 aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg 420 ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg 480 tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc 540 gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat 600 ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc 660 gtgtcgtgcc tgagagcgca gtcgaattcg ccacc atg aag atc ccc aat atc 713 ggc aac gtg atg aat aag ttc gag atc ctg gga gtg gtg gga gag gga 761 gca tac gga gtg gtg ctg aag tgc cgg cac aag gag aca cac gag atc 809 gtg gcc atc aag aag ttt aag gat agc gag gag aat gag gag gtg aag 857 gag aca acc ctg agg gag ctg aag atg ctg cgc aca ctg aag cag gag 905 aac atc gtg gag ctg aag gag gcc ttc cgg aga agg ggc aag ctg tac 953 ctg gtg ttt gag tac gtg gag aag aac atg ctg gag ctg ctg gag gag 1001 atg cct aat ggc gtg ccc cct gag aag gtg aag agc tac atc tat cag 1049 ctg atc aag gcc atc cac tgg tgc cac aag aac gac atc gtg cac agg 1097 gat atc aag cca gag aac ctg ctg atc agc cac aat gac gtg ctg aag 1145 ctg tgc gat ttc ggc ttt gcc cgc aac ctg tcc gag ggc aac aac gcc 1193 aat tac aca gag tac gtg gca acc agg tgg tac cgg tcc cca gag ctg 1241 ctg ctg gga gca cca tac ggc aag agc gtg gac atg tgg tcc gtg ggc 1289 tgc atc ctg ggc gag ctg tct gac ggc cag cct ctg ttc cca ggc gag 1337 agc gag atc gat cag ctg ttt acc atc cag aag gtg ctg ggc cct ctg 1385 cca agc gag cag atg aag ctg ttc tac tcc aac cct agg ttc cac ggc 1433 ctg cgc ttt ccc gcc gtg aat cac cct cag agc ctg gag cgc cgg tat 1481 ctg ggc atc ctg aac tcc gtg ctg ctg gat ctg atg aag aat ctg ctg 1529 aag ctg gac ccc gcc gat agg tac ctg acc gag cag tgt ctg aac cac 1577 ccc aca ttc cag acc cag aga ctg ctg gac agg tcc cct tct cgc aat 1625 cag gcc ggc aag tct aca gcc ctg cag agc cac cac agg agc aac tcc 1673 aag gac atc cag aat ctg agc gtg ggc ctg cct agg gca gac gag ggc 1721 ctg cca gca aac gag tcc ttt ctg aat gga aac ctg gca gga gcc tct 1769 ctg agc cca ctg cac aca aag acc tac cag gcc agc tcc cag cca ggc 1817 tcc aca tct aag gat ctg acc aac aat aac atc cca cac ctg ctg tct 1865 ccc aag gag gcc aag agc aag acc gag ttc gac ttt aat atc gat ccc 1913 aag cct agc gag ggc cca ggc aca aag tat ctg aag agc aac tcc cgg 1961 agc cag cag aat aga cac tcc ttc atg gag tct agc cag tct aag gcc 2009 ggc acc ctg cag ccc aac gag aag cag agc cgc cac tcc tac atc gat 2057 acc atc ccc cag tcc tct agg agc cct tcc tat cgc aca aag gcc aag 2105 agc cac ggc gcc ctg tct gac agc aag tcc gtg tct aat ctg tcc gag 2153 gca agg gca cag atc gca gag ccc agc acc tcc aga tac ttt cct agc 2201 tcc tgt ctg gac ctg aac tct cca aca agc cca aca ccc acc cgg cac 2249 agc gat aca aga acc ctg ctg tct ccc agc ggc aga aat aac agg aac 2297 gag ggc acc ctg gat tct aga agg acc aca acc cgg cac agc aag aca 2345 atg gag gag ctg aag ctg ccc gag cac atg gac tct agc cac tcc cac 2393 tct ctg agc gcc cct cac gag tcc ttc tct tac ggc ctg ggc tat acc 2441 tcc cca ttt tcc tct caa cag cgc ccc cac cgg cac tct atg tac gtg 2489 aca cgg gac aag gtg aga gcc aag ggc ctg gac ggc agc ctg tcc atc 2537 gga cag gga atg gca gca aga gca aac tcc ctg cag ctg ctg tct cct 2585 cag cca gga gag cag ctg cca cca gag atg acc gtg gca cgg agc tcc 2633 gtg aag gag aca agc aga gag ggc acc tct agc ttc cac aca cgg cag 2681 aag tcc gag ggc ggc gtg tat cac gac cct cac tct gac gac ggc acc 2729 gcc cca aag gag aac aga cac ctg tac aac gat cca gtg cct agg agg 2777 gtg ggc tcc ttc tat cgg gtg cca tct ccc cgc cct gac aat agc ttt 2825 cac gag aat aac gtg tcc acc cgg gtg tcc tct ctg ccc tct gag agc 2873 tcc tct ggc aca aac cac tct aag aga cag ccc gcc ttt gac cct tgg 2921 aag agc cca gag aat atc tct cac agc gag cag ctg aag gag aag gag 2969 aag cag ggc ttc ttt agg agc atg aag aag aag aag aag aag agc cag 3017 acc gtg cct aac tcc gac tct ccc gat ctg ctg acc ctg cag aag agc 3065 atc cac agc gcc tcc aca cca agc tcc cgg cct aag gag tgg cgg ccc 3113 gag aag atc tcc gat ctg cag acc cag agc cag cca ctg aag tcc ctg 3161 cgc aag ctg ctg cac ctg tct agc gcc agc aac cac cct gcc tcc tct 3209 gac cca agg ttc cag ccc ctg aca gcc cag cag acc aag aac tct ttt 3257 agc gag atc cgc atc cac cca ctg tcc caa gcc tct gga ggc agc tcc 3305 aac atc agg cag gag cca gca cca aag ggc cgc ccc gcc ctg cag ctg 3353 cct gga cag atg gac ccc gga tgg cac gtg tct agc gtg aca cgg tcc 3401 gcc acc gag gga cca tcc tac tct gag cag ctg gga gca aag agc gga 3449 cct aac gga cac cca tat aac aga acc aat aga tcc agg atg cct aat 3497 ctg aac gac ctg aag gag aca gcc ctg tga tag aagcttatcg ataatcaacc 3550 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 3610 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 3670 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 3730 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3790 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3850 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3910 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 3970 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 4030 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 4090 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgatcgag 4150 gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat 4210 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 4270 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca 4330 gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga 4390 taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact 4450 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg 4510 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg 4570 agcgagcgcg cag 4583 <210> 8 <211> 943 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 8 Met Lys Ile Pro Asn Ile Gly Asn Val Met Asn Lys Phe Glu Ile Leu 1 5 10 15 Gly Val Val Gly Glu Gly Ala Tyr Gly Val Val Leu Lys Cys Arg His 20 25 30 Lys Glu Thr His Glu Ile Val Ala Ile Lys Lys Phe Lys Asp Ser Glu 35 40 45 Glu Asn Glu Glu Val Lys Glu Thr Thr Leu Arg Glu Leu Lys Met Leu 50 55 60 Arg Thr Leu Lys Gln Glu Asn Ile Val Glu Leu Lys Glu Ala Phe Arg 65 70 75 80 Arg Arg Gly Lys Leu Tyr Leu Val Phe Glu Tyr Val Glu Lys Asn Met 85 90 95 Leu Glu Leu Leu Glu Glu Met Pro Asn Gly Val Pro Pro Glu Lys Val 100 105 110 Lys Ser Tyr Ile Tyr Gln Leu Ile Lys Ala Ile His Trp Cys His Lys 115 120 125 Asn Asp Ile Val His Arg Asp Ile Lys Pro Glu Asn Leu Leu Ile Ser 130 135 140 His Asn Asp Val Leu Lys Leu Cys Asp Phe Gly Phe Ala Arg Asn Leu 145 150 155 160 Ser Glu Gly Asn Asn Ala Asn Tyr Thr Glu Tyr Val Ala Thr Arg Trp 165 170 175 Tyr Arg Ser Pro Glu Leu Leu Leu Gly Ala Pro Tyr Gly Lys Ser Val 180 185 190 Asp Met Trp Ser Val Gly Cys Ile Leu Gly Glu Leu Ser Asp Gly Gln 195 200 205 Pro Leu Phe Pro Gly Glu Ser Glu Ile Asp Gln Leu Phe Thr Ile Gln 210 215 220 Lys Val Leu Gly Pro Leu Pro Ser Glu Gln Met Lys Leu Phe Tyr Ser 225 230 235 240 Asn Pro Arg Phe His Gly Leu Arg Phe Pro Ala Val Asn His Pro Gln 245 250 255 Ser Leu Glu Arg Arg Tyr Leu Gly Ile Leu Asn Ser Val Leu Leu Asp 260 265 270 Leu Met Lys Asn Leu Leu Lys Leu Asp Pro Ala Asp Arg Tyr Leu Thr 275 280 285 Glu Gln Cys Leu Asn His Pro Thr Phe Gln Thr Gln Arg Leu Leu Asp 290 295 300 Arg Ser Pro Ser Arg Asn Gln Ala Gly Lys Ser Thr Ala Leu Gln Ser 305 310 315 320 His His Arg Ser Asn Ser Lys Asp Ile Gln Asn Leu Ser Val Gly Leu 325 330 335 Pro Arg Ala Asp Glu Gly Leu Pro Ala Asn Glu Ser Phe Leu Asn Gly 340 345 350 Asn Leu Ala Gly Ala Ser Leu Ser Pro Leu His Thr Lys Thr Tyr Gln 355 360 365 Ala Ser Ser Gln Pro Gly Ser Thr Ser Lys Asp Leu Thr Asn Asn Asn 370 375 380 Ile Pro His Leu Leu Ser Pro Lys Glu Ala Lys Ser Lys Thr Glu Phe 385 390 395 400 Asp Phe Asn Ile Asp Pro Lys Pro Ser Glu Gly Pro Gly Thr Lys Tyr 405 410 415 Leu Lys Ser Asn Ser Arg Ser Gln Gln Asn Arg His Ser Phe Met Glu 420 425 430 Ser Ser Gln Ser Lys Ala Gly Thr Leu Gln Pro Asn Glu Lys Gln Ser 435 440 445 Arg His Ser Tyr Ile Asp Thr Ile Pro Gln Ser Ser Arg Ser Pro Ser 450 455 460 Tyr Arg Thr Lys Ala Lys Ser His Gly Ala Leu Ser Asp Ser Lys Ser 465 470 475 480 Val Ser Asn Leu Ser Glu Ala Arg Ala Gln Ile Ala Glu Pro Ser Thr 485 490 495 Ser Arg Tyr Phe Pro Ser Ser Cys Leu Asp Leu Asn Ser Pro Thr Ser 500 505 510 Pro Thr Pro Thr Arg His Ser Asp Thr Arg Thr Leu Leu Ser Pro Ser 515 520 525 Gly Arg Asn Asn Arg Asn Glu Gly Thr Leu Asp Ser Arg Arg Thr Thr 530 535 540 Thr Arg His Ser Lys Thr Met Glu Glu Leu Lys Leu Pro Glu His Met 545 550 555 560 Asp Ser Ser His Ser His Ser Leu Ser Ala Pro His Glu Ser Phe Ser 565 570 575 Tyr Gly Leu Gly Tyr Thr Ser Pro Phe Ser Ser Gln Gln Arg Pro His 580 585 590 Arg His Ser Met Tyr Val Thr Arg Asp Lys Val Arg Ala Lys Gly Leu 595 600 605 Asp Gly Ser Leu Ser Ile Gly Gln Gly Met Ala Ala Arg Ala Asn Ser 610 615 620 Leu Gln Leu Leu Ser Pro Gln Pro Gly Glu Gln Leu Pro Pro Glu Met 625 630 635 640 Thr Val Ala Arg Ser Ser Val Lys Glu Thr Ser Arg Glu Gly Thr Ser 645 650 655 Ser Phe His Thr Arg Gln Lys Ser Glu Gly Gly Val Tyr His Asp Pro 660 665 670 His Ser Asp Asp Gly Thr Ala Pro Lys Glu Asn Arg His Leu Tyr Asn 675 680 685 Asp Pro Val Pro Arg Arg Val Gly Ser Phe Tyr Arg Val Pro Ser Pro 690 695 700 Arg Pro Asp Asn Ser Phe His Glu Asn Asn Val Ser Thr Arg Val Ser 705 710 715 720 Ser Leu Pro Ser Glu Ser Ser Ser Gly Thr Asn His Ser Lys Arg Gln 725 730 735 Pro Ala Phe Asp Pro Trp Lys Ser Pro Glu Asn Ile Ser His Ser Glu 740 745 750 Gln Leu Lys Glu Lys Glu Lys Gln Gly Phe Phe Arg Ser Met Lys Lys 755 760 765 Light Light Light Light Ser Gln Thr Val Pro Asn Ser Asp Ser Pro Asp Leu 770 775 780 Leu Thr Leu Gln Lys Ser Ile His Ser Ala Ser Thr Pro Ser Ser Arg 785 790 795 800 Pro Lys Glu Trp Arg Pro Glu Lys Ile Ser Asp Leu Gln Thr Gln Ser 805 810 815 Gln Pro Leu Lys Ser Leu Arg Lys Leu Leu His Leu Ser Ser Ala Ser 820 825 830 Asn His Pro Ala Ser Ser Asp Pro Arg Phe Gln Pro Leu Thr Ala Gln 835 840 845 Gln Thr Lys Asn Ser Phe Ser Glu Ile Arg Ile His Pro Leu Ser Gln 850 855 860 Ala Ser Gly Gly Ser Ser Asn Ile Arg Gln Glu Pro Ala Pro Lys Gly 865 870 875 880 Arg Pro Ala Leu Gln Leu Pro Gly Gln Met Asp Pro Gly Trp His Val 885 890 895 Ser Ser Val Thr Arg Ser Ala Thr Glu Gly Pro Ser Tyr Ser Glu Gln 900 905 910 Leu Gly Ala Lys Ser Gly Pro Asn Gly His Pro Tyr Asn Arg Thr Asn 915 920 925 Arg Ser Arg Met Pro Asn Leu Asn Asp Leu Lys Glu Thr Ala Leu 930 935 940 <210> 9 <211> 4706 <212> DNA <213> Artificial sequence <220> <223> Expression cassette <220> <221> repeat_region <222> (1)..(130) <223> 5'ITR <220> <221> misc_feature <222> (130)..(168) <223> AAV2 sequence upstream of 3' ITR <220> <221> promoter <222> (213)..(678) <223> Human synaptic protein <220> <221> misc_feature <222> (690) (695) <223> Kozak <220> <221> CDS <222> (696)...(3653) <223> hCDKL5-4GS <220> <221> misc_feature <222> (3668)..(4255) <223> WRPE <220> <221> polyA_signal <222> (4273)...(4511) <223> SV40 PolyA <220> <221> misc_feature <222> (4539)...(4577) <223> AAV2 sequence upstream of 3' ITR <220> <221> repeat_region <222> (4577)...(4706) <223> 3'ITR <400> 9 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggaagatcc tctagaacta tagctagcat gcctgcagag ggccctgcgt atgagtgcaa 240 gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac 300 ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg 360 aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg 420 ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg 480 tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc 540 gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat 600 ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc 660 gtgtcgtgcc tgagagcgca gtcgaattcg ccacc atg aag atc ccc aat atc 713 ggc aac gtg atg aat aag ttc gag atc ctg gga gtg gtg gga gag gga 761 gca tac gga gtg gtg ctg aag tgc cgg cac aag gag aca cac gag atc 809 gtg gcc atc aag aag ttt aag gat agc gag gag aat gag gag gtg aag 857 gag aca acc ctg agg gag ctg aag andg ctg cgc aca ctg aag cag gag 905 aac atc gtg gag ctg aag gag gcc ttc cgg aga agg ggc aag ctg tac 953 ctg gtg ttt gag tac gtg gag aag aac andg ctg gag ctg ctg gag gag 1001 atg cca aat ggc gtg ccc cct gag aag gtg aag tcc tac atc tat cag 1049 ctg atc aag gcc atc cac tgg tgc cac aag aac gac atc gtg cac agg 1097 gat atc aag ccc gag aac ctg ctg atc tcc cac aat gac gtg ctg aag 1145 ctg tgc gat ttc ggc ttt gcc cgc aac ctg agc gag ggc aac aac gcc 1193 aat tac aca gag tac gtg gca acc agg tgg tac cgg agc cct gag ctg 1241 ctg ctg gga gca cca tac ggc aag tct gtg gac atg tgg agc gtg ggc 1289 tgc atc ctg gga gag ctg tcc gac gga cag cca ctg ttc cct ggc gag 1337 tct gag atc gat cag ctg ttt acc atc cag aag gtg ctg ggc ccc ctg 1385 cct tcc gag cag atg aag ctg ttc tac tct aac cct agg ttc cac ggc 1433 ctg cgc ttt cca gcc gtg aat cac ccc cag tct ctg gag cgc cgg tat 1481 ctg ggc atc ctg aac agc gtg ctg ctg gat ctg atg aag aat ctg ctg 1529 aag ctg gac ccc gcc gat agg tac ctg acc gag cag tgt ctg aac cac 1577 cct aca ttc cag acc cag aga ctg ctg gac agg tcc cca tct cgc aat 1625 cag gcc ggc aag tcc aca gcc ctg cag tct cac cac agg agc aac tcc 1673 aag gac atc cag aat ctg tcc gtg ggc ctg cca agg gca gac gag ggc 1721 ctg cct gcc aac gag agc ttt ctg aat gga aac ctg gca gga gcc tct 1769 ctg agc cca ctg cac aca aag acc tac cag gcc agc tcc cag cct ggc 1817 tcc aca tct aag gac ctg acc aac aat aac atc cct cac ctg ctg agc 1865 cca aag gag gcc aag tcc aag acc gag ttc gac ttt aat atc gat cca 1913 aag cca tcc gag gga cca gga aca aag tat ctg aag agc aac tcc cgg 1961 agc cag cag aat aga cac tct ttc atg gag tct agc cag agc aag gcc 2009 ggc acc ctg cag cct aac gag aag cag agc cgc cac tcc tac atc gat 2057 acc atc cca cag tcc tct agg agc ccc tcc tat cgc aca aag gcc aag 2105 tcc cac ggc gcc ctg tct gac agc aag tcc gtg tct aat ctg agc gag 2153 gca agg gca cag atc gca gag cca agc acc tcc aga tac ttt ccc agc 2201 tcc tgt ctg gac ctg aac tct ccc aca agc cct aca cca acc cgg cac 2249 agc gat aca aga acc ctg ctg tct cct agc ggc aga aat aac agg aac 2297 gag ggc acc ctg gat agc aga agg acc aca acc agg cac tcc aag aca 2345 atg gag gag ctg aag ctg cct gag cac atg gac tct agc cac tcc cac 2393 tct ctg agc gcc cca cac gag tcc ttc tct tac ggc ctg ggc tat acc 2441 agc cct ttt tcc tct caa caa cgc cca cac cgg cac tcc atg tac gtg 2489 aca cgg gac aag gtg aga gcc aag ggc ctg gac ggc agc ctg tcc atc 2537 gga cag gga atg gca gca agg gca aat agc ctg cag ctg ctg tcc cca 2585 cag cct gga gag cag ctg cca cca gag atg acc gtg gca cgg agc tcc 2633 gtg aag gag aca agc aga gag ggc acc tct agc ttc cac aca cgg cag 2681 aag agc gag ggc ggc gtg tat cac gac cca cac tcc gac gac gga acc 2729 gca cct aag gag aac aga cac ctg tac aac gat cca gtg ccc cgc cgg 2777 gtg ggc tct ttc tat cgg gtg cct agc cca cgc ccc gac aac tcc ttt 2825 cac gag aat aac gtg tct acc cgg gtg tcc tct ctg ccc agc gag agc 2873 tcc tct ggc aca aac cac tcc aag aga cag cca gcc ttt gat ccc tgg 2921 aag tct cct gag aat atc tct cac agc gag cag ctg aag gag aag gag 2969 aag cag ggc ttc ttt cgg tcc atg aag aag aag aag aag aag agc cag 3017 aca acc gac agc acc aac ggc gag aat cca agc atc aag aag tcc ctg 3065 ttc ccc ctg ttt aac agc aag aat cac ctg aag cac agc tcc tct ctg 3113 aag aag ctg ccc gtg gtg aca cct cca atg gtg ccc aat tcc gac tct 3161 cct gat ctg ctg acc ctg cag aag tcc atc cac agc gcc tcc aca cca 3209 agc tcc cgg ccc aag gag tgg cgg ccc gag aag atc agc gat ctg cag 3257 acc cag tcc cag ccc ctg aag tct ctg aga aag ctg ctg cac ctg tct 3305 agc gcc tcc aac cac cca gcc tcc tct gac cct cgg ttc cag cca ctg 3353 aca gcc cag cag acc aag aac tct ttt agc gag atc cgc atc cac cca 3401 ctg tcc caa gcc tct gga ggc agc tcc aac atc agg cag gag cct gcc 3449 cca aag ggc cgc cca gcc ctg cag ctg cca gga cag andg gac ccc ggc 3497 tgg cac gtg tct agc gtg aca cgg agc gcc acc gag gga cct tcc tac 3545 tct gag cag ctg gga gca aag tct gga cca aac gga cac cct tat aac 3593 aga acc aat aga agc agg atg cca aat ctg aac gac ctg aag gag aca 3641 gcc ctg tga tag aagcttatcg ataatcaacc tctggattac aaaatttgtg 3693 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 3753 taatgccttt gtatcatgct attgcttccc gtatggcttt catttctcc tccttgtata 3813 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 3873 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 3933 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 3993 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 4053 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg 4113 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 4173 tgctgccggc tctgcggcct cttccgcgtc ttcgcttcg ccctcagacg agtcggatct 4233 ccctttgggc cgcctccccg catcgatacc gtcgatcgag gccgcttcga gcagacatga 4293 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 4353 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 4413 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt 4473 tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat 4533 ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg 4593 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc 4653 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cag 4706 <210> 10 <211> 984 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 10 Met Lys Ile Pro Asn Ile Gly Asn Val Met Asn Lys Phe Glu Ile Leu 1 5 10 15 Gly Val Val Gly Glu Gly Ala Tyr Gly Val Val Leu Lys Cys Arg His 20 25 30 Lys Glu Thr His Glu Ile Val Ala Ile Lys Lys Phe Lys Asp Ser Glu 35 40 45 Glu Asn Glu Glu Val Lys Glu Thr Thr Leu Arg Glu Leu Lys Met Leu 50 55 60 Arg Thr Leu Lys Gln Glu Asn Ile Val Glu Leu Lys Glu Ala Phe Arg 65 70 75 80 Arg Arg Gly Lys Leu Tyr Leu Val Phe Glu Tyr Val Glu Lys Asn Met 85 90 95 Leu Glu Leu Leu Glu Glu Met Pro Asn Gly Val Pro Pro Glu Lys Val 100 105 110 Lys Ser Tyr Ile Tyr Gln Leu Ile Lys Ala Ile His Trp Cys His Lys 115 120 125 Asn Asp Ile Val His Arg Asp Ile Lys Pro Glu Asn Leu Leu Ile Ser 130 135 140 His Asn Asp Val Leu Lys Leu Cys Asp Phe Gly Phe Ala Arg Asn Leu 145 150 155 160 Ser Glu Gly Asn Asn Ala Asn Tyr Thr Glu Tyr Val Ala Thr Arg Trp 165 170 175 Tyr Arg Ser Pro Glu Leu Leu Leu Gly Ala Pro Tyr Gly Lys Ser Val 180 185 190 Asp Met Trp Ser Val Gly Cys Ile Leu Gly Glu Leu Ser Asp Gly Gln 195 200 205 Pro Leu Phe Pro Gly Glu Ser Glu Ile Asp Gln Leu Phe Thr Ile Gln 210 215 220 Lys Val Leu Gly Pro Leu Pro Ser Glu Gln Met Lys Leu Phe Tyr Ser 225 230 235 240 Asn Pro Arg Phe His Gly Leu Arg Phe Pro Ala Val Asn His Pro Gln 245 250 255 Ser Leu Glu Arg Arg Tyr Leu Gly Ile Leu Asn Ser Val Leu Leu Asp 260 265 270 Leu Met Lys Asn Leu Leu Lys Leu Asp Pro Ala Asp Arg Tyr Leu Thr 275 280 285 Glu Gln Cys Leu Asn His Pro Thr Phe Gln Thr Gln Arg Leu Leu Asp 290 295 300 Arg Ser Pro Ser Arg Asn Gln Ala Gly Lys Ser Thr Ala Leu Gln Ser 305 310 315 320 His His Arg Ser Asn Ser Lys Asp Ile Gln Asn Leu Ser Val Gly Leu 325 330 335 Pro Arg Ala Asp Glu Gly Leu Pro Ala Asn Glu Ser Phe Leu Asn Gly 340 345 350 Asn Leu Ala Gly Ala Ser Leu Ser Pro Leu His Thr Lys Thr Tyr Gln 355 360 365 Ala Ser Ser Gln Pro Gly Ser Thr Ser Lys Asp Leu Thr Asn Asn Asn 370 375 380 Ile Pro His Leu Leu Ser Pro Lys Glu Ala Lys Ser Lys Thr Glu Phe 385 390 395 400 Asp Phe Asn Ile Asp Pro Lys Pro Ser Glu Gly Pro Gly Thr Lys Tyr 405 410 415 Leu Lys Ser Asn Ser Arg Ser Gln Gln Asn Arg His Ser Phe Met Glu 420 425 430 Ser Ser Gln Ser Lys Ala Gly Thr Leu Gln Pro Asn Glu Lys Gln Ser 435 440 445 Arg His Ser Tyr Ile Asp Thr Ile Pro Gln Ser Ser Arg Ser Pro Ser 450 455 460 Tyr Arg Thr Lys Ala Lys Ser His Gly Ala Leu Ser Asp Ser Lys Ser 465 470 475 480 Val Ser Asn Leu Ser Glu Ala Arg Ala Gln Ile Ala Glu Pro Ser Thr 485 490 495 Ser Arg Tyr Phe Pro Ser Ser Cys Leu Asp Leu Asn Ser Pro Thr Ser 500 505 510 Pro Thr Pro Thr Arg His Ser Asp Thr Arg Thr Leu Leu Ser Pro Ser 515 520 525 Gly Arg Asn Asn Arg Asn Glu Gly Thr Leu Asp Ser Arg Arg Thr Thr 530 535 540 Thr Arg His Ser Lys Thr Met Glu Glu Leu Lys Leu Pro Glu His Met 545 550 555 560 Asp Ser Ser His Ser His Ser Leu Ser Ala Pro His Glu Ser Phe Ser 565 570 575 Tyr Gly Leu Gly Tyr Thr Ser Pro Phe Ser Ser Gln Gln Arg Pro His 580 585 590 Arg His Ser Met Tyr Val Thr Arg Asp Lys Val Arg Ala Lys Gly Leu 595 600 605 Asp Gly Ser Leu Ser Ile Gly Gln Gly Met Ala Ala Arg Ala Asn Ser 610 615 620 Leu Gln Leu Leu Ser Pro Gln Pro Gly Glu Gln Leu Pro Pro Glu Met 625 630 635 640 Thr Val Ala Arg Ser Ser Val Lys Glu Thr Ser Arg Glu Gly Thr Ser 645 650 655 Ser Phe His Thr Arg Gln Lys Ser Glu Gly Gly Val Tyr His Asp Pro 660 665 670 His Ser Asp Asp Gly Thr Ala Pro Lys Glu Asn Arg His Leu Tyr Asn 675 680 685 Asp Pro Val Pro Arg Arg Val Gly Ser Phe Tyr Arg Val Pro Ser Pro 690 695 700 Arg Pro Asp Asn Ser Phe His Glu Asn Asn Val Ser Thr Arg Val Ser 705 710 715 720 Ser Leu Pro Ser Glu Ser Ser Ser Gly Thr Asn His Ser Lys Arg Gln 725 730 735 Pro Ala Phe Asp Pro Trp Lys Ser Pro Glu Asn Ile Ser His Ser Glu 740 745 750 Gln Leu Lys Glu Lys Glu Lys Gln Gly Phe Phe Arg Ser Met Lys Lys 755 760 765 Light Light Light Light Ser Gln Thr Thr Asp Ser Thr Asn Gly Glu Asn Pro 770 775 780 Ser Ile Lys Lys Ser Leu Phe Pro Leu Phe Asn Ser Lys Asn His Leu 785 790 795 800 Lys His Ser Ser Ser Leu Lys Lys Leu Pro Val Val Thr Pro Pro Met 805 810 815 Val Pro Asn Ser Asp Ser Pro Asp Leu Leu Thr Leu Gln Lys Ser Ile 820 825 830 His Ser Ala Ser Thr Pro Ser Ser Arg Pro Lys Glu Trp Arg Pro Glu 835 840 845 Lys Ile Ser Asp Leu Gln Thr Gln Ser Gln Pro Leu Lys Ser Leu Arg 850 855 860 Lys Leu Leu His Leu Ser Ser Ala Ser Asn His Pro Ala Ser Ser Asp 865 870 875 880 Pro Arg Phe Gln Pro Leu Thr Ala Gln Gln Thr Lys Asn Ser Phe Ser 885 890 895 Glu Ile Arg Ile His Pro Leu Ser Gln Ala Ser Gly Gly Ser Ser Asn 900 905 910 Ile Arg Gln Glu Pro Ala Pro Lys Gly Arg Pro Ala Leu Gln Leu Pro 915 920 925 Gly Gln Met Asp Pro Gly Trp His Val Ser Ser Val Thr Arg Ser Ala 930 935 940 Thr Glu Gly Pro Ser Tyr Ser Glu Gln Leu Gly Ala Lys Ser Gly Pro 945 950 955 960 Asn Gly His Pro Tyr Asn Arg Thr Asn Arg Ser Arg Met Pro Asn Leu 965 970 975 Asn Asp Leu Lys Glu Thr Ala Leu 980 <210> 11 <211> 22 <212> Ms <213> artificial sequence <220> <223> target <400> 11 agtgaattct accagtgcca ta 22 <210> 12 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Target site <400> 12 agcaaaaatg tgctagtgcc aaa 23 <210> 13 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Target sequence <400> 13 agtgtgagtt ctaccattgc caaa 24 <210> 14 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Target sequence <400> 14 agggattcct gggaaaactg gac 23 <210> 15 <211> 16 <212> DNA <213> Artificial sequence <220> <223> spacer <400> 15 atgacttaaa ccaggt 16 <210> 16 <211> 3430 <212> DNA <213> Artificial sequence <220> <223> Transcription variants <400> 16 gggggagttg gaggaggaag accctacagg aagttgagct gtactctcca acaggcgagc 60 acatggtcgt ctcctctctc ccttcaggga gtcatttaat acttcatgat tagaacaaat 120 atgtgaaagt tcccaccaac cagtgagaat ttcttccttc agacggtttt ggatcttact 180 gcacagcttt ctgagaagtt cttttggtgc catgttttgt ggcttgcatc aaaagaggag 240 tttgtcttca tgaagattcc taacattggt aatgtgatga ataaatttga gatccttggg 300 gttgtaggtg aaggagccta tggagttgta cttaaatgca gacacaagga aacacatgaa 360 attgtggcga tcaagaaatt caaggacagt gaagaaaatg aagaagtcaa agaaacgact 420 ttacgagagc ttaaaatgct tcggactctc aagcaggaaa acattgtgga gttgaaggaa 480 gcatttcgtc ggaggggaaa gttgtacttg gtgtttgagt atgttgaaaa aaatatgctc 540 gaattgctgg aagaaatgcc aaatggagtt ccacctgaga aagtaaaaag ctacatctat 600 cagctaatca aggctattca ctggtgccat aagaatgata ttgtccatcg agatataaaa 660 ccagaaaatc tcttaatcag ccacaatgat gtcctaaaac tgtgtgactt tggttttgct 720 cgtaatctgt cagaaggcaa taatgctaat tacacagagt acgttgccac cagatggtat 780 cggtccccag aactcttact tggcgctccc tatggaaagt ccgtggacat gtggtcggtg 840 ggctgtattc ttggggagct tagcgatgga cagcctttat ttcctggaga aagtgaaatt 900 gaccaacttt ttactattca gaaggtgcta ggaccacttc catctgagca gatgaagctt 960 ttctacagta atcctcgctt ccatgggctc cggtttccag ctgttaacca tcctcagtcc 1020 ttggaaagaa gataccttgg aattttgaat agtgttctac ttgacctaat gaagaattta 1080 ctgaagttgg acccagctga cagatacttg acagaacagt gtttgaatca ccctacattt 1140 caaacccaga gacttgga tcgttctcct tcaaggtcag caaaaagaaa accttaccat 1200 gtggaaagca gcacattgtc tatagaaac caagccggca aaagtactgc tttgcagtct 1260 caccacagat ctaacagcaa ggacatccag aacctgagtg taggcctgcc ccgggctgac 1320 gaaggtctcc ctgccaatga aagcttccta aatggaaacc ttgctggagc tagtcttagt 1380 ccactgcaca ccaaaacta ccaagcaagc agccagcctg ggtctaccag caaagatctc 1440 accaacaaca acataccaca ccttcttagc ccaaaagaag ccaagtcaaa aacagagttt 1500 gattttaata ttgacccaaa gccttcagaa ggcccaggga caaagtacct caagtcaaac 1560 agcagatctc agcagaaccg ccactcattc atggaaagct ctcaaagcaa agctgggaca 1620 ctgcagccca atgaaaagca gagtcggcat agctatattg acacattcc ccagtcctct 1680 aggagtccct cctacaggac caaggccaaa agccatgggg cactgagtga ctccaagtct 1740 gtgagcaacc tttctgaagc cagggcccaa attgcggagc ccagtaccag taggtacttc 1800 ccatctagct gcttagactt gaattctccc accagcccaa ccccaccag acacagtgac 1860 acgagaactt tgctcagccc ttctggaaga aataaccgaa atgagggaac gctggactca 1920 cgtcgaacca caaccagaca ttctaagacg atgggagaat tgaagctgcc ggagcacatg 1980 gacagtagcc attcccattc actgtctgca cctcacgaat ctttttctta tggactgggc 2040 tacaccagcc ccttttcttc ccagcaacgt cctcataggc attctatgta tgtgacccgt 2100 gacaaagtga gagccaaggg cttggatgga agcttgagca tagggcaagg gatggcagct 2160 agagccaaca gcctgcaact cttgtcaccc cagcctggag aacagctccc tccagagatg 2220 actgtggcaa gatctcggt caagacc tccagagaag gcacccttc cttccataca 2280 cgccagaagt ctgagggtgg agtgtatcat gacccacact ctgatgatgg cacagccccc 2340 aaagaaaata vakacctata caatgatccct gtgccaagga gagttggtag cttttacaga 2400 gtgccatctc cacgtccaga caatctttc catgaaaata atgtgtcaac tagattttct 2460 tctctaccat cagagagcag ttctggacc aaccactca aagacaacc agcattcgat 2520 ccatggaaaa gtcctgaaaa tattagtcat tcagagcaac tcaggaaaa aggaagcaa 2580 ggatttttca ggtcaatgaa aaagaaaag aaaaatctc aaacagtacc caatccgac 2640 agccctgatc ttctgacgtt gcagaaatcc attcattctg ctagcactcc aagcagcaga 2700 ccaaaggagt ggcgccccga gaagactctca gatctgcaga cccaaagcca gccattaaaa 2760 tcactgcgca agttgttaca tctcttcg gcctcaatc acccggctc ctcagatccc 2820 cgcttccagc ccttaacagc tcaaaacc aaaaattcct tctcagaat tcggattcac 2880 cccctgagcc aggcctctgg cgggagcagc aacatccggc aggaacccgc accgaagggc 2940 aggccagccc tccagctgcc agacggtgga tgtgatggca gaagacagag acaccattct 3000 ggaccccaag atagacgctt catgttaagg acgacagaac aacaaggaga atacttctgc 3060 tgtggtgacc caaagaagcc tcacactccg tgcgtcccaa accgagccct tcatcgtcca 3120 atctccagtc ctgctcccta tccagtactc caggtccgag gcacttccat gtgcccgaca 3180 ctccaggtcc gaggcactga tgctttcagc tgcccaaccc agcaatccgg gttctctttc 3240 ttcgtgagac acgttatgag ggaagccctg attcacaggg cccaggtaaa ccaagctgcg 3300 ctcctgacat accatgagaa tgcggcactg acgggcaagt gacttctgca agcctgcggc 3360 tggtcccaat gccctgaatc acctctctca tggaagaacc aattaacacc aatgaatcaa 3420 ccattaaaaa 3430 <210> 17 <211> 14572 <212> DNA <213> Artificial sequence <220> <223> Transcript variant <400> 17 agttgtctct gccgctgggg aaggtaaagc ggcgacggcg tcctcaggag ctgtggggtc 60 120 tatgtgaaag ttcccaccaa ccagtgaagaa tttcttcctt cagacggttt tggatcttac 180 tgcacagctt tctgagaagt tcttttggtg ccatgttttg tggcttgcat caaaagaga 240 gtttgtcttc atgaagattc ctaacattgg taatgtgatg ataaatttg agatccttgg 300 ggttgtaggt gaaggagcct atggagttgt acttaaatgc agacacaagg aaacacatga 360 aattgtggcg atcaagaaat tcaaggacag tgaaaaaat gaagaagtca aagaaacgac 420 480 agcatttcgt cggaggggaa agttgtactt ggtgtttgag tatgttgaaa aaaatatgct 540 cgaattgctg gaagaaatgc caaatggagt tccacctgag aaagtaaaaa gctacatcta 600 tcagctaatc aaggctattc actggtgcca taagaatgat attgtccatc gagatataaa 660 accagaaaat ctcttaatca gccacaatga tgtcctaaaa ctgtgtgact ttggttttgc 720 tcgtaatctg tcagaaggca ataatgctaa ttacacagag tacgttgcca ccagatggta 780 tcggtcccca gaactcttac ttggcgctcc ctatggaag tccgtggaca tgtggtcggt 840 gggctgtatt cttggggagc ttagcgatgg acagcctttta tttcctggag aaagtgaaat tgaccaactt tttactattc agaaggtgct aggaccactt ccatctgagc agaaggct tttctacagt aatcctcgct tccatgggct ccggtttcca gctgttaacc atcctcagtc cttggaaaga agataccttg gaatttga tagtgttcta cttgacctaa tgaagaattt actgaagttg gacccagctg acagatactt gacagaacag tgtttgaatc accctacatt tcaaacccag agacttctgg atcgttctcc ttcaaggtca gcaaaaagaa aaccttacca tgtggaaagc agcacattgt ctaatagaaa ccaagccggc aaaagtactg ctttgcagtc tcaccacaga tctaacagca aggacatcca gaacctgagt gtaggcctgc cccgggctga cgaaggtctc cctgccaatg aaagcttcct aaatggaaac cttgctggag ctagtcttag tccactgcac accaaaacct accaagcaag cagccagcct gggtctacca gcaaagatct 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 tgattttaat attgacccaa agccttcaga aggcccaggg acaagtacc tcaagtcaaa 1560 cagcagatct cagcagaacc gccactcatt catggaaagc tctcaaagca aagctggggac 1620 actgcagccc aatgaaaagc agagtcggca tagctatatt gacacaattc cccagtcctc 1680 taggagtccc tcctacagga ccaaggccaa aagccatggg gcactgagtg actccaagtc 1740 tgtgagcaac ctttctgaag ccagggccca aattgcggag cccagtacca gtaggtactt 1800 cccatctagc tgcttagact tgaattctcc caccagccca accccacca gacacagtga 1860 cacgagaact ttgctcagcc cttctggaag aaataaccga aatgagggaa cgctggactc 1920 acgtcgaacc acaaccagac attctaagac gatggagaa ttgaagctgc cggagcacat 1980 ggacagtagc cattcccatt cactgtctgc acctcacgaa tcttttctt atggactggg 2040 ctacaccagc cccttttctt cccagcaacg tcctcatagg cattctatgt atgtgacccg 2100 tgacaaagtg agagccaagg gcttggatgg aagcttgagc atagggcaag ggatggcagc 2160 tagagccaac agcctgcaac tcttgtcacc ccagcctgga gaacagctcc ctccagagat 2220 gactgtggca agatcttcgg tcaaagagac ctccagagaa ggcacctctt ccttccatac 2280 acgccagaag tctgagggtg gagtgtatca tgacccacac tctgatgatg gcacagcccc 2340 aaaaaat agaaccctat acaatgatcc tgtgccaagg agagttggta gcttttacag 2400 agtgccatct ccacgtccag acaattcttt ccatgaaaat aatgtgtcaa ctagagtttc 2460 2520 2580 aggatttttc aggtcaatga aaaagaaaaa gaagaaatct caaacagtac ccaattccga 2640 cagccctgat cttctgacgt tgcagaaatc cattcattct gctagcactc caagcagcag 2700 accaaaggag tggcgccccg agaagatctc agatctgcag acccaaagcc agccattaaa 2760 atcactgcgc aagttgttac atctctcttc ggcctcaaat cacccggctt cctcagatcc 2820 ccgcttccag cccttaacag ctcaacaaac caaaaattcc ttctcagaaa ttcggattca 2880 ccccctgagc caggcctctg gcgggagcag caacatccgg caggaacccg caccgaaggg 2940 caggccagcc ctccagctgc caggtcagat ggatcctggt tggcatgtgt cctctgtgac 3000 caggagtgcc acagagggcc cttcctactc tgaacagctg ggtgccaaaa gtgggccaaa 3060 tgggcacccc tataacagaa caaatcgctc acgaatgcca aatctgaatg attaaaaga 3120 gacagccttg taatttgtgc tggtaggggg gaggggtgga cagacaagcc agtggggagg 3180 ggtgggaaag ggtgggctgg gcttggggca tgggccgggc caagtggtga gccaatattt 3240 tcagctttat gaaggtgtgt gcaatattgc atgtgttggg gccgttgagc tcctcgcggc 3300 cacaaatgct agtcagggat cttagagcca caggagttcc ttagggatcg ccactcccca 3360 caggtcttgt gtgagaatag atagagtgtg ccattgagga agaagaaatt cttgccagtt 3420 tctccccttt acattccagc tttagtgcaa tctctgttga acttgggaat gccaggatgt 3480 gtcctggcac tgcaagggat aggaaagtcg tgttgaccga tgcccttact acatattttt 3540 ttctgcctag agtaaatgtg gggtttattg taatccaaga gtatcccttt gaagggttag 3600 cagatgaact gtatgtctta aattgttttc taaacttcca tatttgatag gatttgtaac 3660 attatttat attaatatt gtactgttaa aatcatatct tttatgttat aatgtaaagt 3720 tattttgagc tgtttgaaac attgtgaagc agtgggacag ctacagtagt ttctataaaa 3780 actaaacagt aacatttata tattgcatca ggagtatttt attctatata tatatatat 3840 atatggtaaa tatctgtttt ataaatatat tcatttaaag aaagtatcgt tatgacacta 3900 tctgccatat tttatacat ttgtgatatg aagtgagtat tatacttcta ctaaagggag 3960 ttgaattgtg gctacacgtg actgtaacag tatgaacctt gctcaacttt gaggccccag 4020 cagtagcctc tagacatgat ccttggtagc agacgagatg cagcatctct ttccaaacct 4080 gcaggggaag aaagtcagat aggccagtga gaattccagc aggcctggtt tccgtcctca 4140 cacacttggc tcctgtttgt gtccagggac gttacttgca cacaggggag aatagattga 4200 ggcgttactc gtgggtcttt gtagagagaa tgtgctcttc cacttagcat tagtgtagag 4260 cgtagaccaa agatttgcca gtgaacattc cttgttgtga gatcataaga ggtacctgtc 4320 tgcagatctt gcaaaccatc tctatgctcc tgaatattgt ccactgtccc ggagactctt 4380 ggctgatggg gtgtgagata tatgtgtggc atttctattt ctgaatgcct tttttgctat 4440 tgctctctcc aataccatat ttaaaaggct cctggagcaa ttccagatag ataaaaaatt actctatcca tttttttctt cctggggctg tccacttatg acaagatttt catgcacacc 4560 4620. tgttacgcac acaaccccca tcagaacagg acctatcttc ccctcatctg atctctttca gctcccaagt tactcccaag tatcatcaaa ccctttggcc atcaagtgtt atcctcccat gcctctgggt catccttgag ggaagtcacc tcagcaccat catctatcag tagtag 4740 ttttttggat tagtctctga gcaataatac ttttaaaggt ccctcccacc cctcaaagag ataacttctc agtatttatt acttgtcctc aaattctgag tgtcagcccc tgcatcaagt cagctatgat caaaatgtgc tgttactgtt aaccctcccc cgagattttg agaatcatag ctctgatgat tatgaagatt father aggtcctagt ttccctgaag cttaaattgt gcacatattg catttttata taaatactat aatgtgtatt gattatag ataggtcatt ttaggtgct ttttatttaa ttttaatgag tgtaataggc actaaaatga aactcgactg ggtactatta ctgaaaaaat ttgactcaaa ccaatagttt tgcatgctct ttagttcttt 5160 ttctgtctta cttctttcac cagttcagta ctgtttgggt agctctgaat tctggctttt 5220 ccaggcagct gcttgatgat acaaatgagg tggaccatca gcagttttgc ctggacacta 5280 atgactctaa aagggtgtgt atttaactct tctttttcat actagatctc taccttgtat 5340 tccccccttg caaaccagaa actacacttt ttttctctgg aaagacttct cactgtgcta 5400 tgcgcttagg aactgcacgg tcccgttgcc atgctgtggc attcgaggct cgtcacctag 5460 gggctggttt cccatgttgt catccttgct aacactggga tctcagatgt ttgcagaaca 5520 tttctattca tgacggttct tcaaagaagg gctagaatat ttgccttcta cctagagcaa 5580 agtaaaccta attgttgaag agtcaatacg tactttttgt actttcccag atttgagcca 5640 gaaaatacct acagatatct acaaatgttt tcaaccagtg ttttcgaggt agctctttaa 5700 tcctcttctc agcttttgtc tgttctgact tttcatccaa taagctgtaa gtgaactaca 5760 atctgcattt ccagcatgca attctcttcc tgctcacgga gggggatgca aaagttttct 5820 ctcacaccta atgggccctc tcttagcttt taactgttgt tttcccctgt tgatgaaaaa 5880 ttactgacat cacaaagatt gccatccatg gtagaaagga cttgatttaa agtcagaaaa 5940 atttgccttt tctttaggaa ggggaattca tgacatccat gtcctattat cggccgtaac 6000 ttcctaaaga agaaaaaggg tgaatgaaag cttatgttac ttttatgaaa gtatcactga 6060 tctcctttgg aaaagaaact ctgcttcact gttttctac ctttatttct ctgccatcct 6120 cgttttcct ctctgaagtt ctaaaataccc tttaactgaa ccttgttggt cttgctctaa 6180 aatttagcct ctttctagag cccaagacca aaagggagtg cttcatgctg aggggctcaa 6240 gtgagctgac tctacaactg tggttctcaa ctaggggcag atttacccc aagggtacat 6300 ttggcaatgt ctggggacat ttggttgtca tagctgggg ggaggtgatg gtatgctact 6360 ggcatcaagt ggttagaggc cacagatgct actaaacacc ctgccatgca caggacagcc 6420 ccccaaacaa acagtggtcc agccccaagc actactagtg ctgaggttga gaaaccctgc 6480 cctacaccat cccaagccca ccagtctgca gagcttggct ctggccttgt tctgtccagg 6540 ctacagtact gcaggtgtga tttcttcaac atcacattct cttaagagtc ttcaaccact 6600 atgcctgaat ccttggaatc ctattgttcg tttccattgt cttgggcctg ctaaaaggtg 6660 catactttgg aaggacca gagttacttt ataggtgttg gagaactaa attctaggtt 6720 agcatcctta gaaaatcttt catagagcca tgaggcttat atttagaagc aagcaacagc 6780 ctggcagatt ggcatgattt ttaccaactg ctcaaaggca tccgtggctt tcagctgtgt 6840 ctcccgaaag aaaatgtctt tgtttttat tcaagtcatt taatagctct ttacaaatat 6900 tagcatatgg cagaccaatt agaagcaaaa aagtccgtat tggtggttgt gatgtggctg 6960 ttatagaagt atttgtgctg tatgctttgg ttaaatctgt tttaaaacat gctttaagat 7020 tcaccttacg cagttgtact ttaattctaa gctcagagct atgctgtcag tctctagtca 7080 cgccatgtat tataagtat gttgtggttg tagagttagc cagtttagca tgttcctaat 7140 ctgagaatta gtggactata ctaggaaatg ctatcccatt ttccctttcc agcccctctt 7200 7260 ccatttattt tcttctagcc atgtattatt gagaatgact catagtacaa ctttttttca 7320 aaggccagag gattactttg caagtgtgta gaaagatctg tctatttctg ttcacctggg 7380 acctggtggt gactgaactc tttcagcaag ggcaatggct caatgtgatt cggtgctgga 7440 gctcagctgg gactcagtaa atctgcacgt tctctgctgg tcagaacatg tttcctggag 7500 agcattgctt ttctatctag aagttcgtta cctcctgcat gatagtggga tattgagatg 7560 tgggttctaa gatccacaga ctcatacttt tgtgaacttt ggaatgtggt agggtacact 7620 gaaccaagtc agaaaatagt gaaatatttc cttgcctcct tcagttcatc actaagaaag 7680 tgtgtaggca gagaaaactt acttatggtt tgaagaacca caattgtttt tgaaagggga 7740 catagaaaag acagtacaaa ggttgtaagt gtcctgtcgc taattcccag ggccagaatc 7800 tcacaagaag atcctctcaa agacttaaaa gcctggctta caaaaataac cagaaaccaa 7860 acttgtaagc agttacaatt tcttcctttt gcttcttgac cagctgagat acagaatatc 7920 ttgaggtctg gttcagctga tgaaaaattc tgtctcagaa acgatgagta agaaaccata 7980 caagaaaaag aagtccctca tctatctgca tacacgcaat gtgggagaag tggggtggct 8040 aggaaactct gggcctgctg cccccttcga ttcttttttt ttttctttgt gaattgaatg 8100 tacgatacaa atggtaggcc ttcatgtgag ccagttacta catgaatctt catttcccac 8160 agtggtttgt tcattcatca gcgttaggct tggtcctggc tccacctttc tcctctccgg 8220 gcactgaccc cacctttccg tgtatttact gtaggctatt aaatatgatc atgacccgcc 8280 ttgcattttc attcatcacc tgtttatgcc caaatttaaa ggaagtttgt ctcattttgc 8340 cagaaaaaaa ttgtaatagt cggcacgctg gatttgtagg gccagcaaaa ttgcggcagt 8400 gaaactagtt tcacttctaa agcccttcat ttcccacaag gttaagctct cgaaacccca 8460 tttgatcctt ggttcctatt tcgatcctcc tttggaatct gaaaatcggt ctccatgttg 8520 tatgcagatt agaagttgcc ttgtttgtta ctcttccaac acagggtatc agggagaaag 8580 aggccttatc tgttcctcca tcccccctgt tttgacagac tgctaagaat tcctcaggac 8640 ttcctttggt tggggatttt actttcccaa aagtctgatc tgatttcttt caggggtaga 8700 caagcttgtc ctagtgctct gcttcaggtc ttatcagaag aaacccagga atagaaaagg 8760 tagatgcctt gacttttgtc cctgttgtgg ggactaaagt gttttttgcc agaattgtca 8820 aaagctccgg ttcaaactct gtagagtttc atggaaaaac aaaacaaaac aaaaaagatg 8880 cttatcgtcc cggagaatgt gtaagtaagt tctccagcac ggcttaggtt ttccaaaatg 8940 gaaagcaaag cttgcagaca acctgttttc tctagagaca cacaatgatt ctggccctca 9000 gtctgtttcc ctccccttgt ttactctttg gtcaccgatc gagtcaggct agaggggtaa 9060 tacggtgatt aaaagaagta aattctcact gtaaacttgg attgattaac agaaacaatt 9120 aaaccaaaca aaatcattgc cccaaatttc tatctccaag aattgctttg tgccattttg 9180 aattcaggta gttattctgt atatacttag ctaactattc agagcttctt caatctctag 9240 taagcttgaa ggtggtgtct gggaagacac agggcagggt gacatttaga ctctgttcat 9300 tcttaaaaat agggataagc cagaatgatg tgacatctag tatgtggtag atgaagaatc 9360 agtggaaatt cttaattgca cagactttat aatccattat ataaagtgta attccatact 9420 ttttactgtg gcaagggtgt gatgtgatg ttaatcttt taatttttga atccaactg 9480 aatttaaagt gttgaatact taaatcctca caagtgaatt atgaaccatt tgtaagtgt 9540 ttcttaact ctttgtatca tgtgttgta catcttatca agttttctct ggcatgtaat 9600 tccattctaa acttatgtaa atttctatt gttgctgtaa atattacaca atatctatt 9660 ccgtgataac cttcattatc agcatgaat ggttaatttt tactgagcac aatgtatttt 9720 tgaatggatc ttcccaatg tctttaataa atgcagat tgcactgac tgatgtgact 9780 gccaggccgt cccaatcttg agcactgtgg tccttcgtgt ttgaactgcg aacagctcca 9840 ttttagttcc ttctgggaa gaagaatgc attcgaatttc aaatgaggga tgatggggct 9900 ttgaggttgt atcagtttg agacagaaat taatgtaaaa ctaggcaaat cctgtaactt 9960 aatgatgttg atctttgttt tcctatttat aaagctttt tccccttacg gcattttgaa 10020 aactttggtt tgttttac tgagtgttg tcaccttgc acgtcgctag acacctcacgt 10080 ggtagtttga ggaggatggg aagagaggcc aatcttgtgc taagtgctat ggagaaagca 10140 aagatgcaca gcttaataag gttttttga ttctgagga gaggtgggca agctttcct 10200 gtaaagggcc agatggtaaa tattgggggc tctgtggccc atcagttccc tgtcacatt 10260 actcagctct gcctttgtat tgcagaggct gcatggataa tatgtaatc agtaagtgtg 10320 gctgtggtcc cgtaaaactt tacttataga aacaggccat agtttacta cccgttttag 10380 gccctcagtt tcatcattta gggtacacc aaccgagtac atgttcaatc aggaggaaa 10440 gaacttagct ttgtgataa cttatagatg aaatattccc cagttttctt tctctttcta 10500 aacaaagggt aggagctgaa agtcagtgct ctccattctt aaagtcctc tgtcaccac 10560 ctcttcactc tatctcctta agtgacatgg aaacttgtgg cctgtgcttt caactataat 10620 cttaccaaga ctgaatgaaa gtatttggtg cttatacttt ttaatgttca tagcacttac 10680 tatggccagg cacaatgccc aagtgttttg ctttattatt attattatta ttattat 10740 tattattatt attattatt tcacacac ccagcgaac aggtactatt atttacccct 10800 tttgtaaat gaggaaacag atttccatac ttgacattaa cctgcccaag gtcaccagt 10860 ttggaagtgg cagggccagg atttgaagcc gatatgttc attgtaaagc ccatataccc 10920 ttaaaagtca ctgcacgtcc acgtatgta atgtggtcac aatcagtatt gtttctcttt 10980 agtaattcag atttattca gtaactccaa gttcaatt ttcaaaaca gtttccaaca 11040 caggttatat taaaaacttt gcctgaagtt tactctaggt gcagaaataa agacaactta 11100 gtcaagtgaa ggaagggta acagacttag aaatggctat ctacactgat ggaataagta 11160 tactaattag agcaggtaac aagtcccac tactaac atattctct ctaaaagctc 11220 acaacaattt atgtgccacc ttccacccc ttcccagca gatggtaatt gatgcaattt 11280 gagaatagaa cactcattg aaacaagtca ctggaggtcc tctgggatg cagcttaaaa 11340 gcccaggatc aggccaggtg tggtggctca cgcctgtaat cccagcactg tgggagactg 11400 agacggggcag atcacctgaa gtcaggagtt cgagaccagc ctggccaata tagagaacc 11460 ccgtctgtac taaaaataca aaaattaggt ctgggcggtg gctcacgccc gtaatcctag 11520 cactttggga ggccaaggtg ggtggatcac gaggtcagga gttcgagacc agcctggcca 11580 acatggtgaa accccgtctc tactaaaaat acaaaaattg ggtgtggtgg tacatgcctg 11640 taatcccagc tgcttaggag gctgaggcag gagaatcact tgaacccaaa aggcagaggt 11700 tgcagtgagc cgagatcacg ccactgcact ccagcctggc aacagagcga gactctgtct 11760 caaaaaaaaa aaaaaaaaaa tacaaaaatt agccgggaat ggtggtgaat gtctataatc 11820 ccagccactc gggaggctga ggcaggagaa tcacttgaac ccaaaaggca gaggttgcag 11880 tgagccaaga tcgcaccact gcactcccac ctggacgaca gagcgagacg ccatctcaaa 11940 aataaataaa taaataaatc cctaggatat agagtccgct tgcctgggtg gagtctttggc 12000 tccatctcct acatctcatg tgacttgaag caaggcactt cactatgcct cagtttcctt 12060 atctttaaat ggaggtaatc acagtaccta cctcagtgtt gttgtgaaga tttaatgaga 12120 tataacctg cagagcactt gagagtctca agaaatatga gctcttataa cattattgct 12180 attgtaaaac attgctatta cttctgtccc aatttggcag tttcaggaaa ttgagaagga 12240 atgtcactga atctttaatg ccagttacac agagctcagt aggtgtccgc acagcccttt 12300 agatgcacgt agcctatttg atcttcgtat cagccctgta aggttgggct taagaatgac 12360 ccagcatgga agtactcttg ggaccacagg ggaagcaaca gtccttgtaa gctgtttgtc 12420 agtgggcctt agcaaccaag ccccaagtgg acagaaagat ggggttctgt gagcatccat 12480 tccaagggtc actttagggg tgcaatcttc tgggctctga gaatggattc acagcagcat 12540 ctctatctgg aaatacctttt gctttgtcca agtaacctcc tcccacacag agtacccgaa 12600 tcagctcaga ttttttttcc ttaatattgg caacacttaa tgctacttct ttgttcgtaa 12660 catttcattt aattatttaa aaatctgcaa atgttgtgta tattttaccg caatttttaa 12720 acaaactcca gtgtagaccg tgctctagca tttctaaaaa taaaagtaaa tatgttaaaa 12780 taaggtaaga gtgagttttt cctacactat gtataattttt taattgaaag tttcccccac 12840 aaaagaaatg atacttggaa gcatagtttg tgtgtttgac agcaagattc tattaggctt 12900 cagatttccc tcataaaag ttttagtttt tatacattct gtcatgaatt gatgaagta 12960 aaatatgc tgttgtaagc aaattatctg cagataaatg tcttatgaat cgtatatctt 13020 actgtttatt aaattctgaa gtattgagca gttccaatg tgaatccca gctggctct 13080 ttgcaatgat ggacaagtta gttatatattc attggaat tcaggaacc cagaatagcc 13140 aaaacaatcc cgagagcaa agttggagga ctcacatgcc ccaatttca aacttac 13200 aaagctaca taatgagaa agtgaggta taggattgg agtccagaa taaaccctct 13260 cacatttatg gtcaatgtt tttgacaag ggtgccaaga tcattcaatg ggggaaagaa 13320 tagtcttttc agcaatagt ggtgggacaa ctggatatcc acatgcaaaa gatgacgtt 13380 ggactcctat ttcaccat atacaaaaat taactcaaa tggacaaag atctaactgt 13440 aagaggtaga actataaaac tcatagaga aaacataggg gtaaatctc atgaccttgg 13500 atttggcagt ggtttctcag attgacacc aaaggcacaa gcaagagaa aaataattg 13560 gacttcatca atattattaa atttgtgct tagaggact ctacgagag ggttaaaaag 13620 atccacagaa tgggagaaaa tatttgcaaa tcatatccaa taagggaatt agaatataca 13680 aagaattctt aaactcagta agttgtaagc cgatttaaaa tgggcagagg atctgaaaaa 13740 cctttatcca aagatgtaca aatggccagt aaacacatga aaagatgctc aacatcatta 13800 gtcactaggg aaatgcaaat caaaaccaca gttgagatgc aacttcacac attatgggta 13860 tagtcaaaga tggacagtaa caagtgttgc tgaggatgtg gagaaagtgg aagcctccta 13920 cattgctggt ggaaatgtaa aatgatgcag ccacttggga taatagttcc tccaaagatt 13980 aaacaagagt gactgtgtgt cccagcagtt ttactcctaa gtatataccc aagataaatg 14040 aaaacacaca tccgcacaaa aacttataca caaatgttca tagcagcatt attcataata 14100 gctaaagagt ggaaacccaa atatccatca atggatgaat ggataaataa caatgtgctc 14160 ttgacatgat actgaacaat gatgttcagc aataaaaaag agtgacaagt gacatagtga 14220 tacacatgtt ataacgtgga tgaactttaa aacactatgc taagtgaaag aagccagtca 14280 cgaaatacca catactatat gatttcattt atgtagaacg tccagaaggc caatctacag 14340 agacagtcga ttcgtggttg cagagggctt ggggtggccg atgggttggg gagtgttggc 14400 tgatgggtgc aggatttctc tttggggtga taaactttct aaaattgatc tagtgataat 14460 ggttgcacaa ctctgaatat accaaaagtc atcaaattgt acactttaaa tggatacaaa 14520 ttgtatgtga acttaatctc aataaagccg tgacaaaagt ttccaacatg aa 14572 <210> 18 <211> 3434 <212> DNA <213> Artificial sequence <220> <223> Transcript variant <400> 18 attagttgtc tctgccgctg gggaaggtaa agcggcgacg gcgtcctcag gagctgtggg 60 gtcccctgct agaagtgggg gactcggcgg gggagtcatt taatacttca tgattagaac 120 aaatatgtga aagttcccac caaccagtga gaatttcttc cttcagacgg ttttggatct 180 tactgcacag ctttctgaga agttcttttg gtgccatgtt ttgtggcttg catcaaaaga 240 ggagtttgtc ttcatgaaga ttcctaacat tggtaatgtg atgaataaat ttgagatcct 300 tggggttgta ggtgaaggag cctatggagt tgtacttaaa tgcagacaca aggaaacaca 360 tgaaattgtg gcgatcaaga aattcaagga cagtgaagaa aatgaagaag tcaaagaaac 420 gactttacga gagcttaaaa tgcttcggac tctcaagcag gaaaacattg tggagttgaa 480 ggaagcattt cgtcggaggg gaaagttgta cttggtgttt gagtatgttg aaaaaaatat 540 gctcgaattg ctggaagaaa tgccaaatgg agttccacct gagaaagtaa aaagctacat 600 ctatcagcta atcaaggcta ttcactggtg ccataagaat gatattgtcc atcgagatat 660 aaaaccagaa aatctcttaa tcagccacaa tgatgtccta aaactgtgtg actttggtttt 720 tgctcgtaat ctgtcagaag gcaataatgc taattacaca gagtacgttg ccaccagatg 780 gtatcggtcc ccagaactct tacttggcgc tccctatgga aagtccgtgg acatgtggtc 840 ggtgggctgt attcttgggg agcttagcga tggacagcct ttatttcctg gagaaagtga 900 aattgaccaa cttttacta ttcagaaggt gctaggacca cttccatctg agcagatgaa 960 gctttctac agtaatcctc gcttccatgg gctccggttt ccagctgtta accatcctca 1020 gtccttggaa agaagatacc ttggaattttt gaatagtgtt ctacttgacc taatgaagaa 1080 tttactgaag ttggacccag ctgacagata cttgacagaa cagtgtttga atcaccctac 1140 atttcaaacc cagagacttc tggatcgttc tccttcaagg tcagcaaaaa gaaaacctta 1200 ccatgtggaa agcagcacat tgtctaatag aaaccaagcc ggcaaaagta ctgctttgca 1260 gtctcaccac agatctaaca gcaaggacat ccagaacctg agtgtaggcc tgccccgggc 1320 tgacgaaggt ctccctgcca atgaaagctt cctaaatgga aaccttgctg gagctagtct 1380 tagtccactg cacaccaaaa cctaccaagc aagcagccag cctgggcta ccagcaaaga 1440 tctcaccaac aacaacatac cacaccttct tagcccaaaa gaagccaagt caaaaacaga 1500 gttgatttt aatattgacc caaagccttc agaaggccca gggacaaagt acctcaagtc 1560 aaacagcaga tctcagcaga accgccactc attcatggaa agctctcaaa gcaaagctgg 1620 gacactgcag cccaatgaaa agcagagtcg gcatagctat attgacacaa ttccccagtc 1680 ctctaggagt ccctcctaca ggaccaaggc caaaagccat ggggcactga gtgactccaa 1740 gtctgtgagc aacctttctg aagccagggc ccaaattgcg gagcccagta ccagtaggta cttcccatct agctgcttag acttgaattc tcccaccagc ccaaccccca ccagacacag tgacacgaga actttgctca gcccttctgg aagaatac cgaatgagg gaacgctgga ctcacgtcga accacaacca gacattctaa gacgatggag gattgaagc tgccggagca catggacagt agccattccc attcactgtc tgcacctcac gaatcttttt cttatggact gggctacacc agcccctttt cttcccagca acgtcctcat aggcattcta tgtatgtgac ccgtgacaaa gtgagagcca agggcttgga tggaagcttg agcatagggc aagggatggc agctagagcc aacagcctgc aactcttgtc accccagcct ggagaacagc tccctccaga gatgactgtg gcaagatctt cggtcaaaga gacctccaga gaaggcacct cttccttcca 2340. tacacgccag aagtctgagg gtggagtgta tcatgaccca cactctgatg atggcacagc ccccaaaga aatagacacc tatacaatga tcctgtgcca aggagagttg gtagctttta cagagtgcca tctccacgtc cagacaattc tttccatgaa fathergtgt caactagagt ttcttctcta ccatcagaga gcagttctgg aaccaaccac tcaaaaagac aaccagcatt 2520 cgatccatgg aaaagtcctg aaaatattag tcattcagag caactcaagg aaaaagagaa 2580 gcaaggattt ttcaggtcaa tgaaaaagaa aaagaagaaa tctcaaacag tacccaattc 2640 cgacagccct gatcttctga cgttgcagaa atccattcat tctgctagca ctccaagcag 2700 cagaccaaag gagtggcgcc ccgagaagat ctcagatctg cagacccaaa gccagccatt 2760 aaaatcactg cgcaagttgt tacatctctc ttcggcctca aatcacccgg cttcctcaga 2820 tccccgcttc cagcccttaa cagctcaaca aaccaaaaat tccttctcag aaattcggat 2880 tcaccccctg agccaggcct ctggcgggag cagcaacatc cggcaggaac ccgcaccgaa 2940 gggcaggcca gccctccagc tgccagacgg tggatgtgat ggcagaagac agagacacca 3000 ttctggaccc caagatagac gcttcatgtt aaggacgaca gaacaacaag gagaatactt 3060 ctgctgtggt gacccaaaga agcctcacac tccgtgcgtc ccaaaccgag cccttcatcg 3120 tccaatctcc agtcctgctc cctatccagt actccaggtc cgaggcactt ccatgtgccc 3180 gacactccag gtccgaggca ctgatgcttt cagctgccca acccagcaat ccgggttctc 3240 tttcttcgtg agacacgtta tgagggaagc cctgattcac agggcccagg taaaccaagc 3300 tgcgctcctg acataccatg agaatgcggc actgacgggc aagtgacttc tgcaagcctg 3360 cggctggtcc caatgccctg aatcacctct ctcatggaag aaccaattaa caccaatgaa 3420 tcaaccatta aaaa 3434 <210> 19 <211> 1030 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence <400> 19 Met Lys Ile Pro Asn Ile Gly Asn Val Met Asn Lys Phe Glu Ile Leu 1 5 10 15 Gly Val Val Gly Glu Gly Ala Tyr Gly Val Val Leu Lys Cys Arg His 20 25 30 Lys Glu Thr His Glu Ile Val Ala Ile Lys Lys Phe Lys Asp Ser Glu 35 40 45 Glu Asn Glu Glu Val Lys Glu Thr Thr Leu Arg Glu Leu Lys Met Leu 50 55 60 Arg Thr Leu Lys Gln Glu Asn Ile Val Glu Leu Lys Glu Ala Phe Arg 65 70 75 80 Arg Arg Gly Lys Leu Tyr Leu Val Phe Glu Tyr Val Glu Lys Asn Met 85 90 95 Leu Glu Leu Leu Glu Glu Met Pro Asn Gly Val Pro Pro Glu Lys Val 100 105 110 Lys Ser Tyr Ile Tyr Gln Leu Ile Lys Ala Ile His Trp Cys His Lys 115 120 125 Asn Asp Ile Val His Arg Asp Ile Lys Pro Glu Asn Leu Leu Ile Ser 130 135 140 His Asn Asp Val Leu Lys Leu Cys Asp Phe Gly Phe Ala Arg Asn Leu 145 150 155 160 Ser Glu Gly Asn Asn Ala Asn Tyr Thr Glu Tyr Val Ala Thr Arg Trp 165 170 175 Tyr Arg Ser Pro Glu Leu Leu Leu Gly Ala Pro Tyr Gly Lys Ser Val 180 185 190 Asp Met Trp Ser Val Gly Cys Ile Leu Gly Glu Leu Ser Asp Gly Gln 195 200 205 Pro Leu Phe Pro Gly Glu Ser Glu Ile Asp Gln Leu Phe Thr Ile Gln 210 215 220 Lys Val Leu Gly Pro Leu Pro Ser Glu Gln Met Lys Leu Phe Tyr Ser 225 230 235 240 Asn Pro Arg Phe His Gly Leu Arg Phe Pro Ala Val Asn His Pro Gln 245 250 255 Ser Leu Glu Arg Arg Tyr Leu Gly Ile Leu Asn Ser Val Leu Leu Asp 260 265 270 Leu Met Lys Asn Leu Leu Lys Leu Asp Pro Ala Asp Arg Tyr Leu Thr 275 280 285 Glu Gln Cys Leu Asn His Pro Thr Phe Gln Thr Gln Arg Leu Leu Asp 290 295 300 Arg Ser Pro Ser Arg Ser Ala Lys Arg Lys Pro Tyr His Val Glu Ser 305 310 315 320 Ser Thr Leu Ser Asn Arg Asn Gln Ala Gly Lys Ser Thr Ala Leu Gln 325 330 335 Ser His His Arg Ser Asn Ser Lys Asp Ile Gln Asn Leu Ser Val Gly 340 345 350 Leu Pro Arg Ala Asp Glu Gly Leu Pro Ala Asn Glu Ser Phe Leu Asn 355 360 365 Gly Asn Leu Ala Gly Ala Ser Leu Ser Pro Leu His Thr Lys Thr Tyr 370 375 380 Gln Ala Ser Ser Gln Pro Gly Ser Thr Ser Lys Asp Leu Thr Asn Asn 385 390 395 400 Asn Ile Pro His Leu Leu Ser Pro Lys Glu Ala Lys Ser Lys Thr Glu 405 410 415 Phe Asp Phe Asn Ile Asp Pro Lys Pro Ser Glu Gly Pro Gly Thr Lys 420 425 430 Taurus Leu Lys Ser Asn Ser Arg Ser Gln Gln Asn Arg His Ser Phe Met 435 440 445 Glu Ser Ser Gln Ser Lys Ala Gly Thr Leu Gln Pro Asn Glu Lys Gln 450 455 460 Ser Arg His Ser Tyr Ile Asp Thr Ile Pro Gln Ser Ser Arg Ser Pro 465 470 475 480 Ser Tyr Arg Thr Lys Ala Lys Ser His Gly Ala Leu Ser Asp Ser Lys 485 490 495 Ser Val Ser Asn Leu Ser Glu Ala Arg Ala Gln Ile Ala Glu Pro Ser 500 505 510 Thr Ser Arg Tyr Phe Pro Ser Ser Cys Leu Asp Leu Asn Ser Pro Thr 515 520 525 Ser Pro Thr Pro Thr Arg His Ser Asp Thr Arg Thr Leu Leu Ser Pro 530 535 540 Ser Gly Arg Asn Asn Arg Asn Glu Gly Thr Leu Asp Ser Arg Arg Thr 545 550 555 560 Thr Thr Arg His Ser Lys Thr Met Glu Glu Leu Lys Leu Pro Glu His 565 570 575 Met Asp Ser Ser His Ser His Ser Leu Ser Ala Pro His Glu Ser Phe 580 585 590 Ser Tyr Gly Leu Gly Tyr Thr Ser Pro Phe Ser Ser Gln Gln Arg Pro 595 600 605 His Arg His Ser Met Tyr Val Thr Arg Asp Lys Val Arg Ala Lys Gly 610 615 620 Leu Asp Gly Ser Leu Ser Ile Gly Gln Gly Met Ala Ala Arg Ala Asn 625 630 635 640 Ser Leu Gln Leu Leu Ser Pro Gln Pro Gly Glu Gln Leu Pro Pro Glu 645 650 655 Met Thr Val Ala Arg Ser Ser Val Lys Glu Thr Ser Arg Glu Gly Thr 660 665 670 Ser Ser Phe His Thr Arg Gln Lys Ser Glu Gly Gly Val Tyr His Asp 675 680 685 Pro His Ser Asp Asp Gly Thr Ala Pro Lys Glu Asn Arg His Leu Tyr 690 695 700 Asn Asp Pro Val Pro Arg Arg Val Gly Ser Phe Tyr Arg Val Pro Ser 705 710 715 720 Pro Arg Pro Asp Asn Ser Phe His Glu Asn Asn Val Ser Thr Arg Val 725 730 735 Ser Ser Leu Pro Ser Glu Ser Ser Ser Gly Thr Asn His Ser Lys Arg 740 745 750 Gln Pro Ala Phe Asp Pro Trp Lys Ser Pro Glu Asn Ile Ser His Ser 755 760 765 Glu Gln Leu Lys Glu Lys Glu Lys Gln Gly Phe Phe Arg Ser Met Lys 770 775 780 Light Light Light Light Light Ser Gln Thr Val Pro Asn Ser Asp Ser Pro Asp 785 790 795 800 Leu Leu Thr Leu Gln Lys Ser Ile His Ser Ala Ser Thr Pro Ser Ser 805 810 815 Arg Pro Lys Glu Trp Arg Pro Glu Lys Ile Ser Asp Leu Gln Thr Gln 820 825 830 Ser Gln Pro Leu Lys Ser Leu Arg Lys Leu Leu His Leu Ser Ser Ala 835 840 845 Ser Asn His Pro Ala Ser Ser Asp Pro Arg Phe Gln Pro Leu Thr Ala 850 855 860 Gln Gln Thr Lys Asn Ser Phe Ser Glu Ile Arg Ile His Pro Leu Ser 865 870 875 880 Gln Ala Ser Gly Gly Ser Ser Asn Ile Arg Gln Glu Pro Ala Pro Lys 885 890 895 Gly Arg Pro Ala Leu Gln Leu Pro Asp Gly Gly Cys Asp Gly Arg Arg 900 905 910 Gln Arg His His Ser Gly Pro Gln Asp Arg Arg Phe Met Leu Arg Thr 915 920 925 Thr Glu Gln Gln Gly Glu Tyr Phe Cys Cys Gly Asp Pro Lys Lys Pro 930 935 940 His Thr Pro Cys Val Pro Asn Arg Ala Leu His Arg Pro Ile Ser Ser 945 950 955 960 Pro Ala Pro Tyr Pro Val Leu Gln Val Arg Gly Thr Ser Met Cys Pro 965 970 975 Thr Leu Gln Val Arg Gly Thr Asp Ala Phe Ser Cys Pro Thr Gln Gln 980 985 990 Ser Gly Phe Ser Phe Phe Val Arg His Val Met Arg Glu Ala Leu Ile 995 1000 1005 His Arg Ala Gln Val Asn Gln Ala Ala Leu Leu Thr Tyr His Glu 1010 1015 1020 Asn Ala Ala Leu Thr Gly Lys 1025 1030 <210> 20 <211> 960 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence <400> 20 Met Lys Ile Pro Asn Ile Gly Asn Val Met Asn Lys Phe Glu Ile Leu 1 5 10 15 Gly Val Val Gly Glu Gly Ala Tyr Gly Val Val Leu Lys Cys Arg His 20 25 30 Lys Glu Thr His Glu Ile Val Ala Ile Lys Lys Phe Lys Asp Ser Glu 35 40 45 Glu Asn Glu Glu Val Lys Glu Thr Thr Leu Arg Glu Leu Lys Met Leu 50 55 60 Arg Thr Leu Lys Gln Glu Asn Ile Val Glu Leu Lys Glu Ala Phe Arg 65 70 75 80 Arg Arg Gly Lys Leu Tyr Leu Val Phe Glu Tyr Val Glu Lys Asn Met 85 90 95 Leu Glu Leu Leu Glu Glu Met Pro Asn Gly Val Pro Pro Glu Lys Val 100 105 110 Lys Ser Tyr Ile Tyr Gln Leu Ile Lys Ala Ile His Trp Cys His Lys 115 120 125 Asn Asp Ile Val His Arg Asp Ile Lys Pro Glu Asn Leu Leu Ile Ser 130 135 140 His Asn Asp Val Leu Lys Leu Cys Asp Phe Gly Phe Ala Arg Asn Leu 145 150 155 160 Ser Glu Gly Asn Asn Ala Asn Tyr Thr Glu Tyr Val Ala Thr Arg Trp 165 170 175 Tyr Arg Ser Pro Glu Leu Leu Leu Gly Ala Pro Tyr Gly Lys Ser Val 180 185 190 Asp Met Trp Ser Val Gly Cys Ile Leu Gly Glu Leu Ser Asp Gly Gln 195 200 205 Pro Leu Phe Pro Gly Glu Ser Glu Ile Asp Gln Leu Phe Thr Ile Gln 210 215 220 Lys Val Leu Gly Pro Leu Pro Ser Glu Gln Met Lys Leu Phe Tyr Ser 225 230 235 240 Asn Pro Arg Phe His Gly Leu Arg Phe Pro Ala Val Asn His Pro Gln 245 250 255 Ser Leu Glu Arg Arg Tyr Leu Gly Ile Leu Asn Ser Val Leu Leu Asp 260 265 270 Leu Met Lys Asn Leu Leu Lys Leu Asp Pro Ala Asp Arg Tyr Leu Thr 275 280 285 Glu Gln Cys Leu Asn His Pro Thr Phe Gln Thr Gln Arg Leu Leu Asp 290 295 300 Arg Ser Pro Ser Arg Ser Ala Lys Arg Lys Pro Tyr His Val Glu Ser 305 310 315 320 Ser Thr Leu Ser Asn Arg Asn Gln Ala Gly Lys Ser Thr Ala Leu Gln 325 330 335 Ser His His Arg Ser Asn Ser Lys Asp Ile Gln Asn Leu Ser Val Gly 340 345 350 Leu Pro Arg Ala Asp Glu Gly Leu Pro Ala Asn Glu Ser Phe Leu Asn 355 360 365 Gly Asn Leu Ala Gly Ala Ser Leu Ser Pro Leu His Thr Lys Thr Tyr 370 375 380 Gln Ala Ser Ser Gln Pro Gly Ser Thr Ser Lys Asp Leu Thr Asn Asn 385 390 395 400 Asn Ile Pro His Leu Leu Ser Pro Lys Glu Ala Lys Ser Lys Thr Glu 405 410 415 Phe Asp Phe Asn Ile Asp Pro Lys Pro Ser Glu Gly Pro Gly Thr Lys 420 425 430 Tyr Leu Lys Ser Asn Ser Arg Ser Gln Gln Asn Arg His Ser Phe Met 435 440 445 Glu Ser Ser Gln Ser Lys Ala Gly Thr Leu Gln Pro Asn Glu Lys Gln 450 455 460 Ser Arg His Ser Tyr Ile Asp Thr Ile Pro Gln Ser Ser Arg Ser Pro 465 470 475 480 Ser Tyr Arg Thr Lys Ala Lys Ser His Gly Ala Leu Ser Asp Ser Lys 485 490 495 Ser Val Ser Asn Leu Ser Glu Ala Arg Ala Gln Ile Ala Glu Pro Ser 500 505 510 Thr Ser Arg Tyr Phe Pro Ser Ser Cys Leu Asp Leu Asn Ser Pro Thr 515 520 525 Ser Pro Thr Pro Thr Arg His Ser Asp Thr Arg Thr Leu Leu Ser Pro 530 535 540 Ser Gly Arg Asn Asn Arg Asn Glu Gly Thr Leu Asp Ser Arg Arg Thr 545 550 555 560 Thr Thr Arg His Ser Lys Thr Met Glu Glu Leu Lys Leu Pro Glu His 565 570 575 Met Asp Ser Ser His Ser His Ser Leu Ser Ala Pro His Glu Ser Phe 580 585 590 Ser Tyr Gly Leu Gly Tyr Thr Ser Pro Phe Ser Ser Gln Gln Arg Pro 595 600 605 His Arg His Ser Met Tyr Val Thr Arg Asp Lys Val Arg Ala Lys Gly 610 615 620 Leu Asp Gly Ser Leu Ser Ile Gly Gln Gly Met Ala Ala Arg Ala Asn 625 630 635 640 Ser Leu Gln Leu Leu Ser Pro Gln Pro Gly Glu Gln Leu Pro Pro Glu 645 650 655 Met Thr Val Ala Arg Ser Ser Val Lys Glu Thr Ser Arg Glu Gly Thr 660 665 670 Ser Ser Phe His Thr Arg Gln Lys Ser Glu Gly Gly Val Tyr His Asp 675 680 685 Pro His Ser Asp Asp Gly Thr Ala Pro Lys Glu Asn Arg His Leu Tyr 690 695 700 Asn Asp Pro Val Pro Arg Arg Val Gly Ser Phe Tyr Arg Val Pro Ser 705 710 715 720 Pro Arg Pro Asp Asn Ser Phe His Glu Asn Asn Val Ser Thr Arg Val 725 730 735 Ser Ser Leu Pro Ser Glu Ser Ser Ser Gly Thr Asn His Ser Lys Arg 740 745 750 Gln Pro Ala Phe Asp Pro Trp Lys Ser Pro Glu Asn Ile Ser His Ser 755 760 765 Glu Gln Leu Lys Glu Lys Glu Lys Gln Gly Phe Phe Arg Ser Met Lys 770 775 780 Light Light Light Light Light Ser Gln Thr Val Pro Asn Ser Asp Ser Pro Asp 785 790 795 800 Leu Leu Thr Leu Gln Lys Ser Ile His Ser Ala Ser Thr Pro Ser Ser 805 810 815 Arg Pro Lys Glu Trp Arg Pro Glu Lys Ile Ser Asp Leu Gln Thr Gln 820 825 830 Ser Gln Pro Leu Lys Ser Leu Arg Lys Leu Leu His Leu Ser Ser Ala 835 840 845 Ser Asn His Pro Ala Ser Ser Asp Pro Arg Phe Gln Pro Leu Thr Ala 850 855 860 Gln Gln Thr Lys Asn Ser Phe Ser Glu Ile Arg Ile His Pro Leu Ser 865 870 875 880 Gln Ala Ser Gly Gly Ser Ser Asn Ile Arg Gln Glu Pro Ala Pro Lys 885 890 895 Gly Arg Pro Ala Leu Gln Leu Pro Gly Gln Met Asp Pro Gly Trp His 900 905 910 Val Ser Ser Val Thr Arg Ser Ala Thr Glu Gly Pro Ser Tyr Ser Glu 915 920 925 Gln Leu Gly Ala Lys Ser Gly Pro Asn Gly His Pro Tyr Asn Arg Thr 930 935 940 Asn Arg Ser Arg Met Pro Asn Leu Asn Asp Leu Lys Glu Thr Ala Leu 945 950 955 960 <210> 21 <211> 1030 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence <400> 21 Met Lys Ile Pro Asn Ile Gly Asn Val Met Asn Lys Phe Glu Ile Leu 1 5 10 15 Gly Val Val Gly Glu Gly Ala Tyr Gly Val Val Leu Lys Cys Arg His 20 25 30 Lys Glu Thr His Glu Ile Val Ala Ile Lys Lys Phe Lys Asp Ser Glu 35 40 45 Glu Asn Glu Glu Val Lys Glu Thr Thr Leu Arg Glu Leu Lys Met Leu 50 55 60 Arg Thr Leu Lys Gln Glu Asn Ile Val Glu Leu Lys Glu Ala Phe Arg 65 70 75 80 Arg Arg Gly Lys Leu Tyr Leu Val Phe Glu Tyr Val Glu Lys Asn Met 85 90 95 Leu Glu Leu Leu Glu Glu Met Pro Asn Gly Val Pro Pro Glu Lys Val 100 105 110 Lys Ser Tyr Ile Tyr Gln Leu Ile Lys Ala Ile His Trp Cys His Lys 115 120 125 Asn Asp Ile Val His Arg Asp Ile Lys Pro Glu Asn Leu Leu Ile Ser 130 135 140 His Asn Asp Val Leu Lys Leu Cys Asp Phe Gly Phe Ala Arg Asn Leu 145 150 155 160 Ser Glu Gly Asn Asn Ala Asn Tyr Thr Glu Tyr Val Ala Thr Arg Trp 165 170 175 Tyr Arg Ser Pro Glu Leu Leu Leu Gly Ala Pro Tyr Gly Lys Ser Val 180 185 190 Asp Met Trp Ser Val Gly Cys Ile Leu Gly Glu Leu Ser Asp Gly Gln 195 200 205 Pro Leu Phe Pro Gly Glu Ser Glu Ile Asp Gln Leu Phe Thr Ile Gln 210 215 220 Lys Val Leu Gly Pro Leu Pro Ser Glu Gln Met Lys Leu Phe Tyr Ser 225 230 235 240 Asn Pro Arg Phe His Gly Leu Arg Phe Pro Ala Val Asn His Pro Gln 245 250 255 Ser Leu Glu Arg Arg Tyr Leu Gly Ile Leu Asn Ser Val Leu Leu Asp 260 265 270 Leu Met Lys Asn Leu Leu Lys Leu Asp Pro Ala Asp Arg Tyr Leu Thr 275 280 285 Glu Gln Cys Leu Asn His Pro Thr Phe Gln Thr Gln Arg Leu Leu Asp 290 295 300 Arg Ser Pro Ser Arg Ser Ala Lys Arg Lys Pro Tyr His Val Glu Ser 305 310 315 320 Ser Thr Leu Ser Asn Arg Asn Gln Ala Gly Lys Ser Thr Ala Leu Gln 325 330 335 Ser His His Arg Ser Asn Ser Lys Asp Ile Gln Asn Leu Ser Val Gly 340 345 350 Leu Pro Arg Ala Asp Glu Gly Leu Pro Ala Asn Glu Ser Phe Leu Asn 355 360 365 Gly Asn Leu Ala Gly Ala Ser Leu Ser Pro Leu His Thr Lys Thr Tyr 370 375 380 Gln Ala Ser Ser Gln Pro Gly Ser Thr Ser Lys Asp Leu Thr Asn Asn 385 390 395 400 Asn Ile Pro His Leu Leu Ser Pro Lys Glu Ala Lys Ser Lys Thr Glu 405 410 415 Phe Asp Phe Asn Ile Asp Pro Lys Pro Ser Glu Gly Pro Gly Thr Lys 420 425 430 Tyr Leu Lys Ser Asn Ser Arg Ser Gln Gln Asn Arg His Ser Phe Met 435 440 445 Glu Ser Ser Gln Ser Lys Ala Gly Thr Leu Gln Pro Asn Glu Lys Gln 450 455 460 Ser Arg His Ser Tyr Ile Asp Thr Ile Pro Gln Ser Ser Arg Ser Pro 465 470 475 480 Ser Tyr Arg Thr Lys Ala Lys Ser His Gly Ala Leu Ser Asp Ser Lys 485 490 495 Ser Val Ser Asn Leu Ser Glu Ala Arg Ala Gln Ile Ala Glu Pro Ser 500 505 510 Thr Ser Arg Tyr Phe Pro Ser Ser Cys Leu Asp Leu Asn Ser Pro Thr 515 520 525 Ser Pro Thr Pro Thr Arg His Ser Asp Thr Arg Thr Leu Leu Ser Pro 530 535 540 Ser Gly Arg Asn Asn Arg Asn Glu Gly Thr Leu Asp Ser Arg Arg Thr 545 550 555 560 Thr Thr Arg His Ser Lys Thr Met Glu Glu Leu Lys Leu Pro Glu His 565 570 575 Met Asp Ser Ser His Ser His Ser Leu Ser Ala Pro His Glu Ser Phe 580 585 590 Ser Tyr Gly Leu Gly Tyr Thr Ser Pro Phe Ser Ser Gln Gln Arg Pro 595 600 605 His Arg His Ser Met Tyr Val Thr Arg Asp Lys Val Arg Ala Lys Gly 610 615 620 Leu Asp Gly Ser Leu Ser Ile Gly Gln Gly Met Ala Ala Arg Ala Asn 625 630 635 640 Ser Leu Gln Leu Leu Ser Pro Gln Pro Gly Glu Gln Leu Pro Pro Glu 645 650 655 Met Thr Val Ala Arg Ser Ser Val Lys Glu Thr Ser Arg Glu Gly Thr 660 665 670 Ser Ser Phe His Thr Arg Gln Lys Ser Glu Gly Gly Val Tyr His Asp 675 680 685 Pro His Ser Asp Asp Gly Thr Ala Pro Lys Glu Asn Arg His Leu Tyr 690 695 700 Asn Asp Pro Val Pro Arg Arg Val Gly Ser Phe Tyr Arg Val Pro Ser 705 710 715 720 Pro Arg Pro Asp Asn Ser Phe His Glu Asn Asn Val Ser Thr Arg Val 725 730 735 Ser Ser Leu Pro Ser Glu Ser Ser Ser Gly Thr Asn His Ser Lys Arg 740 745 750 Gln Pro Ala Phe Asp Pro Trp Lys Ser Pro Glu Asn Ile Ser His Ser 755 760 765 Glu Gln Leu Lys Glu Lys Glu Lys Gln Gly Phe Phe Arg Ser Met Lys 770 775 780 Light Light Light Light Light Ser Gln Thr Val Pro Asn Ser Asp Ser Pro Asp 785 790 795 800 Leu Leu Thr Leu Gln Lys Ser Ile His Ser Ala Ser Thr Pro Ser Ser 805 810 815 Arg Pro Lys Glu Trp Arg Pro Glu Lys Ile Ser Asp Leu Gln Thr Gln 820 825 830 Ser Gln Pro Leu Lys Ser Leu Arg Lys Leu Leu His Leu Ser Ser Ala 835 840 845 Ser Asn His Pro Ala Ser Ser Asp Pro Arg Phe Gln Pro Leu Thr Ala 850 855 860 Gln Gln Thr Lys Asn Ser Phe Ser Glu Ile Arg Ile His Pro Leu Ser 865 870 875 880 Gln Ala Ser Gly Gly Ser Ser Asn Ile Arg Gln Glu Pro Ala Pro Lys 885 890 895 Gly Arg Pro Ala Leu Gln Leu Pro Asp Gly Gly Cys Asp Gly Arg Arg 900 905 910 Gln Arg His His Ser Gly Pro Gln Asp Arg Arg Phe Met Leu Arg Thr 915 920 925 Thr Glu Gln Gln Gly Glu Tyr Phe Cys Cys Gly Asp Pro Lys Lys Pro 930 935 940 His Thr Pro Cys Val Pro Asn Arg Ala Leu His Arg Pro Ile Ser Ser 945 950 955 960 Pro Ala Pro Tyr Pro Val Leu Gln Val Arg Gly Thr Ser Met Cys Pro 965 970 975 Thr Leu Gln Val Arg Gly Thr Asp Ala Phe Ser Cys Pro Thr Gln Gln 980 985 990 Ser Gly Phe Ser Phe Phe Val Arg His Val Met Arg Glu Ala Leu Ile 995 1000 1005 His Arg Ala Gln Val Asn Gln Ala Ala Leu Leu Thr Tyr His Glu 1010 1015 1020 Asn Ala Ala Leu Thr Gly Lys 1025 1030 <210> 22 <211> 2883 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence <400> 22 atgaagatcc ccaacatcgg caacgtgatg aacaagttcg agatcctggg cgttgtcgga 60 gaaggcgctt atggcgtggt gctgaagtgc agacacaaag aaacccacga gatcgtggcc 120 atcaagaagt tcaaggacag cgaagagaac gaggaagtga aagagacaac cctgcgcgag 180 ctgaagatgc tgagaaccct gaagcaagag aacatcgtgg aactgaaaga ggccttccgg 240 cggagaggca agctgtacct ggtgttcgag tacgtggaaa agaacatgct cgagctgctg 300 gaagagatgc ccaatggcgt gccacctgag aaagtgaagt cctacatcta ccagctgatc 360 aaggccatcc actggtgcca caagaacgac atcgtgcaca gagacatcaa gcccgagaac 420 ctgctgatct cccacaacga cgtgctgaag ctgtgcgact tcggcttcgc cagaaatctg 480 agcgagggca acaacgccaa ctacaccgag tatgtggcca ccagatggta cagaagccct 540 gaactgctgc tgggagcccc ttacggcaag tctgtggata tgtggtccgt gggctgtatc 600 ctgggagagc tgtctgatgg ccaacctctg tttcctggcg agtccgagat cgaccagctg 660 ttcacaatcc agaaggtgct gggccctctg cctagcgagc agatgaagct gttctacagc 720 aaccccagat tccacggcct gagattccct gccgtgaatc accctcagag cctggaaaga 780 agatacctgg gcatcctgaa cagcgtgctg ctggacctga tgaagaatct gctgaaactg 840 gaccccgccg acagatacct gaccgagcag tgtctgaacc atcctacctt tcagacccag 900 agactgctgg atagaagccc cagcagaagc gccaagagaa agccctacca cgtggaaagc 960 agcaccctga gcaatagaaa ccaggccgga aagagcactg ccctgcagag ccaccacaga 1020 agcaacagca aggacatcca gaacctgagc gtgggcctgc ctagagctga tgaaggactg 1080 cctgccaacg agagcttcct gaatggaaat ctggctggcg cctctctgag ccctctgcac 1140 acaaagacat accaggccag cagccagcct ggcagcacaa gcaaggacct gaccaacaac 1200 aacatccctc atctgctgag ccccaaagag gccaagagca agaccgagtt cgacttcaac 1260 atcgacccca agcctagcga aggccctggc accaagtacc tgaagtccaa ctccagaagc 1320 cagcagaacc ggcactcctt catggaaagc tctcagagca aggccggcac actgcagcct 1380 aacgagaagc agcagaca cagctacatc gacacaatcc ctcagtcctc tcggagcccc 1440 agctacagaa caaaggccaa atctcacggc gccctgagcg atagcaagag cgtgtccaat 1500 ctgtccgagg ctagagcca gatcgccgag cctagcacca gcagatactt ccctagcagc 1560 tgcctggatc tgaacagccc tacaagcccc acacctacca ggcacagcga taccagaaca 1620 ctgctgtccc caagcggccg gaacaacaga aatgagggca ccctggacag cagacggacc 1680 accacaagac acagcaagac catggaagaa ctgaagctgc ccgagcacat ggactctagc 1740 cactctcatt ctctgagcgc ccctcacgag tcctttagct acggcctggg ctacacaagc 1800 ccattttcca gtcagcagcg gccccacaga cactctatgt acgtgaccag acaaaagtc 1860 cgcgccaaag gcctggatgg ctctctgtct attggacaag gcatggccgc cagagccaat 1920 agcctgcaac ttctgtctcc tcagcctggc gaacagctgc ctcctgaaat gacagtggcc 1980 cggtccagcg tgaaagaaac cagcagagag ggcacctcca gcttccacac cagacagaaa 2040 agcgaaggcg gcgtgtacca cgatcctcac tctgatgatg gcacagcccc taagagaac 2100 agacacctgt acaacgaccc cgtgcctaga agagtgggct ccttctacag agtgccttct 2160 cctagacctg acaactcctt ccacgagaac aacgtgtcaa cccgggtgtc cagcctgcct 2220 tctgagagca gctctggcac aaaccactcc aagaggcagc ccgccttcga tccttggaag 2280 tcccctgaga atatcagcca ctccgagcag ctgaaagaga aagagaagca gggctttttc 2340 2400 ctcctgacac tgcagaagtc catccacagc gccagcacac ctagcagcag acctaaagag 2460 tggcggccag agaagatcag cgacctgcaa acacagagcc agcctctgaa gtccctgagg 2520 aagctgctgc atctgtcctc cgcctctaat caccctgcca gcagcgatcc cagatttcag 2580 ccactgacag cccagcagac caagaactcc ttcagcgaga tccggattca cccactgtct 2640 caggccagcg gcggcagctc taatatcaga caagagcccg ctcctaaggg cagacctgct 2700 ctccaactgc ctggacagat ggatcctggc tggcacgtgt cctctgtgac aagatctgcc 2760 accgagggac ctagctattc tgagcagctg ggcgccaagt ctggccccaa tggacacccc 2820 tacaaccgga ccaatcggag cagaatgccc aacctgaacg acctcaaaga aacagccctc 2880 tga 2883 <210> 23 <211> 466 <212> DNA <213> Artificial sequence <220> <223> Promoter <400> 23 ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg 60 gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca 120 aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180 actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg 240 ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc 300 cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg 360 cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc 420 tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcg 466 <210> 24 <211> 3009 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence <400> 24 atgaagatcc ccaatatcgg caacgtgatg aataagttcg agatcctggg agtggtggga 60 gagggagcat acggcgtggt gctgaagtgc agacacaagg agacacacga gatcgtggcc 120 atcaagaagt ttaaggatag cgaggagaat gaggaggtga aggagacaac cctgcgcgag 180 ctgaagatgc tgcggacact gaagcaggag aacatcgtgg agctgaagga ggccttccgg 240 agaaggggca agctgtacct ggtgtttgag tacgtggaga agaacatgct ggagctgctg 300 gaggagatgc ctaatggcgt gccccctgag aaggtgaaga gctacatcta tcagctgatc 360 aaggccatcc actggtgcca caagaacgac atcgtgcacc gcgatatcaa gccagagaac 420 ctgctgatca gccacaatga cgtgctgaag ctgtgcgatt tcggctttgc ccggaacctg 480 tccgagggca acaacgccaa ttacacagag tacgtggcaa ccaggtggta ccggtcccct 540 gagctgctgc tgggagcacc atacggcaag tctgtggaca tgtggagcgt gggctgcatc 600 ctgggagagc tgtccgacgg acagccactg ttccctggcg agtctgagat cgatcagctg 660 tttaccatcc agaaggtgct gggccccctg ccttccgagc agatgaagct gttctactct 720 aacccaaggt tccacggcct gaggtttcca gccgtgaatc acccccagtc tctggagcgc 780 cggtatctgg gcatcctgaa cagcgtgctg ctggatctga tgaagaatct gctgaagctg 840 gaccctgccg atcgctacct gaccgagcag tgtctgaatc acccaacatt tcagacccag 900 agactgctgg acaggagccc ctcccgctct gccaagagga agccttatca cgtggagagc 960 tccaccctga gcaatcgcaa ccaggccggc aagtccacag ccctgcagtc tcaccaccgg 1020 agcaactcca aggacatcca gaatctgagc gtgggcctgc caagggcaga cgagggcctg 1080 cctgccaacg agtccttcct gaatggaaac ctggcaggag cctctctgag cccactgcac 1140 acaaagacct accaggcctc tagccagcct ggctccacat ctaaggacct gaccaacaat 1200 aacatccctc acctgctgag cccaaaggag gccaagtcca agaccgagtt cgactttaat 1260 atcgatccaa agcccagcga gggcccaggc acaaagtatc tgaagagcaa ctccagatct 1320 cagcagaata ggcactcttt catggagtcc tctcagagca aggccggcac cctgcagccc 1380 aacgagaagc agagcaggca ctcctacatc gataccatcc cacagagctc ccgcagcccc 1440 tcctatcgga caaggccaa gagccacggc gccctgtctg acagcaagtc cgtgtctaat 1500 ctgtccgagg caagagcaca gatcgcagag ccaagcacct ccaggtactt tccctcttcc 1560 tgtctggacc tgaactctcc aacaagccct acaccaacca gacacagca tacaaggacc 1620 ctgctgtctc ccagcggcag aataacagg aacgagggca ccctggatag cagaaggacc 1680 aaaccagac actccaagac aatggaggag ctgaagctgc ccgagcacat ggactcctct 1740 cactccact ctctgagcgc ccctcacgag tccttctctt acggcctggg ctataccagc 1800 ccttttagct cccagcagcg cccacacagg cactccatgt acgtgacaag agacaaggtg 1860 agggccaagg gcctggacgg cagcctgtcc atcggacagg gaatggcagc aagggcaaat 1920 agcctgcagc tgctgtcccc acagcctgga gagcagctgc caccagagat gaccgtggcc 1980 agatctagcg tgaaggagac aagcaggagag ggcacctcct ctttccacac aagacagaag 2040 agcgagggcg gcgtgtatca cgacccacac tccgacgacg gaaccgcacc caaggaac 2100 aggcacctgt acaacgatcc agtgccccgc cgggtgggct ctttctatcg ggtgcctagc 2160 ccacggcccg acaactcctt tcacgagaat aacgtgtcta ccagagtgag ctccctgcct 2220 agcgagtcta gctccggcac aaaccactcc aagaggcagc cagcctttga tccctggaag 2280 2340 cgcagcatga agaagaagaa gaagaagagc cagacaaccg acagcaccaa cggcgagaat 2400 cctagcatca agaagtccct gttcccactg tttaactcca agaatcacct gaagcactct 2460 agctccctga agaagctgcc cgtggtgaca cctccaatgg tgcccaattc cgactctcct 2520 gatctgctga ccctgcagaa gagcatccac agcgcctcca caccatctag cagacccaag 2580 gagtggaggc cagaagagat ctccgatctg cagacccagt cccagcccct gaagtctctg 2640 cggaagctgc tgcacctgtc ctctgccagc aaccaccctg caagctccga ccctaggttc 2700 cagccactga cagcccagca gaccaagaac tcttttagcg agatcagaat ccacccactg 2760 tcccaggcct ctggaggctc tagcaacatc cgccaggagc ctgccccaaa gggccggcca 2820 gccctgcagc tgccaggaca gatggacccc ggctggcacg tgtcctctgt gacaagatcc 2880 gccaccgagg gaccatccta ctctgagcag ctgggagcaa agtctggacc aaacggacac 2940 ccttataaca ggaccaatag atccaggatg cctaatctga acgacctgaa ggagacagcc 3000 ctgtgatag 3009 <210> 25 <211> 2835 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid <400> 25 atgaagatcc ccaatatcgg caacgtgatg aataagttcg agatcctggg agtggtggga 60 gagggagcat acggagtggt gctgaagtgc cggcacaagg agacacacga gatcgtggcc 120 atcaagaagt ttaaggatag cgaggagaat gaggaggtga aggagacaac cctgagggag 180 ctgaagatgc tgcgcacact gaagcaggag aacatcgtgg agctgaagga ggccttccgg 240 agaaggggca agctgtacct ggtgtttgag tacgtggaga agaacatgct ggagctgctg 300 gaggagatgc ctaatggcgt gccccctgag aaggtgaaga gctacatcta tcagctgatc 360 aaggccatcc actggtgcca caagaacgac atcgtgcaca gggatatcaa gccagagaac 420 ctgctgatca gccacaatga cgtgctgaag ctgtgcgatt tcggctttgc ccgcaacctg 480 tccgagggca acaacgccaa ttacacagag tacgtggcaa ccaggtggta ccggtcccca 540 gagctgctgc tgggagcacc atacggcaag agcgtggaca tgtggtccgt gggctgcatc 600 ctgggcgagc tgtctgacgg ccagcctctg ttcccaggcg agagcgagat cgatcagctg 660 tttaccatcc agaaggtgct gggccctctg ccaagcgagc agatgaagct gttctactcc 720 aaccctaggt tccacggcct gcgctttccc gccgtgaatc accctcagag cctggagcgc 780 cggtatctgg gcatcctgaa ctccgtgctg ctggatctga tgaagaatct gctgaagctg 840 gaccccgccg ataggtacct gaccgagcag tgtctgaacc accccacatt ccagacccag 900 agactgctgg acaggtcccc ttctcgcaat caggccggca agtctacagc cctgcagagc 960 caccacagga gcaactccaa ggacatccag aatctgagcg tgggcctgcc tagggcagac 1020 gagggcctgc cagcaaacga gtcctttctg aatggaaacc tggcaggagc ctctctgagc 1080 ccactgcaca caaagaccta ccaggccagc tcccagccag gctccacatc taaggatctg 1140 accaacaata acatcccaca cctgctgtct cccaaggagg ccaagagcaa gaccgagttc 1200 gactttaata tcgatcccaa gcctagcgag ggcccaggca caaagtatct gaagagcaac 1260 tcccggagcc agcagaatag acactccttc atggagtcta gccagtctaa ggccggcacc 1320 ctgcagccca acgagaagca gagccgccac tcctacatcg ataccatccc ccagtcctct 1380 aggagccctt cctatcgcac aaaggccaag agccacggcg ccctgtctga cagcaagtcc 1440 gtgtctaatc tgtccgaggc aagggcacag atcgcagagc ccagcacctc cagatacttt 1500 cctagctcct gtctggacct gaactctcca acaagcccaa cacccacccg gcacagcgat 1560 acaagaaccc tgctgtctcc cagcggcaga aataacagga acgagggcac cctggattct 1620 agaaggacca caacccggca cagcaagaca atggaggagc tgaagctgcc cgagcacatg 1680 gactctagcc actcccactc tctgagcgcc cctcacgagt ccttctctta cggcctgggc 1740 tatacctccc cattttcctc tcaacagcgc ccccaccggc actctatgta cgtgacacgg 1800 gacaaggtga gagccaaggg cctggacgg agcctgtcca tcggacaggg aatggcagca 1860 agagcaaact ccctgcagct gctgtctcct cagccaggag agcagctgcc accagagatg 1920 accgtggcac ggagctccgt gaaggaca agcagagagg gcaccttag cttccacaca 1980 cggcagaagt ccgagggcgg cgtgtatcac gaccctcact ctgacgacgg caccgcccca 2040 aaggaaca gacacctgta caacgatcca gtgcctagga gggtgggctc cttctatcgg 2100 gtgccatctc cccgccctga aatagcttt cacgagataa acgtgtccac ccgggtgtcc 2160 tctctgccct ctgagagctc ctctggcaca aaccactcta agagacagcc cgcctttgac 2220 ccttgggaaga gcccagagaa tatctctcac agcgagcagc tgaaggaga ggagaagcag 2280 ggcttcttta ggagcatgaa gaagagaag aagaagagcc agaccgtgcc taactccgac 2340 tctcccgatc tgctgaccct gcagaagagc atccacagcg cctccacacc aagctcccgg 2400 cctaaggagt ggcggcccga gaagatctcc gatctgcaga cccagagcca gccactgaag 2460 tccctgcgca agctgctgca cctgtctagc gccagcaacc accctgcctc ctctgaccca 2520 aggttccagc ccctgacagc ccagcagacc aagaactctt ttagcgagat ccgcatccac 2580 ccactgtccc aagcctctgg aggcagctcc aacatcaggc aggagccagc accaaagggc 2640 cgccccgccc tgcagctgcc tggacagatg gaccccggat ggcacgtgtc tagcgtgaca 2700 cggtccgcca ccgagggacc atcctactct gagcagctgg gagcaaagag cggacctaac 2760 ggacacccat ataacagaac caatagatcc aggatgccta atctgaacga cctgaaggag 2820 acagccctgt gatag 2835 <210> 26 <211> 2958 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence <400> 26 atgaagatcc ccaatatcgg caacgtgatg aataagttcg agatcctggg agtggtggga 60 gagggagcat acggagtggt gctgaagtgc cggcacaagg agacacacga gatcgtggcc 120 atcaagaagt ttaaggatag cgaggagaat gaggaggtga aggagacaac cctgagggag 180 ctgaagatgc tgcgcacact gaagcaggag aacatcgtgg agctgaagga ggccttccgg 240 agaaggggca agctgtacct ggtgtttgag tacgtggaga agaacatgct ggagctgctg 300 gaggagatgc caaatggcgt gccccctgag aaggtgaagt cctacatcta tcagctgatc 360 aaggccatcc actggtgcca caagaacgac atcgtgcaca gggatatcaa gcccgagaac 420 ctgctgatct cccacaatga cgtgctgaag ctgtgcgatt tcggctttgc ccgcaacctg 480 agcgagggca acaacgccaa ttacacagag tacgtggcaa ccaggtggta ccggagccct 540 gagctgctgc tgggagcacc atacggcaag tctgtggaca tgtggagcgt gggctgcatc 600 ctgggagagc tgtccgacgg acagccactg ttccctggcg agtctgagat cgatcagctg 660 tttaccatcc agaaggtgct gggccccctg ccttccgagc agatgaagct gttctactct 720 aaccctaggt tccacggcct gcgctttcca gccgtgaatc acccccagtc tctggagcgc 780 cggtatctgg gcatcctgaa cagcgtgctg ctggatctga tgaagaatct gctgaagctg 840 gaccccgccg ataggtacct gaccgagcag tgtctgaacc accctacatt ccagacccag 900 agactgctgg acaggtcccc atctcgcaat caggccggca agtccacagc cctgcagtct 960 caccacagga gcaactccaa ggacatccag aatctgtccg tgggcctgcc aagggcagac 1020 gagggcctgc ctgccaacga gagctttctg aatggaaacc tggcaggagc ctctctgagc 1080 ccactgcaca caaagaccta ccaggccagc tcccagcctg gctccacatc taaggacctg 1140 accaacaata acatccctca cctgctgagc ccaaaggagg ccaagtccaa gaccgagttc 1200 gactttaata tcgatccaaa gccatccgag ggaccaggaa caaagtatct gaagagcaac 1260 tcccggagcc agcagaatag acactctttc atggagtcta gccagagcaa ggccggcacc 1320 ctgcagccta acgagaagca gagccgccac tcctacatcg ataccatccc acagtcctct 1380 aggagcccct cctatcgcac aaaggccaag tcccacggcg ccctgtctga cagcaagtcc 1440 gtgtctaatc tgagcgaggc aagggcacag atcgcagagc caagcacctc cagatacttt 1500 cccagctcct gtctggacct gaactctccc acaagcccta caccaacccg gcacagcgat 1560 acaagaaccc tgctgtctcc tagcggcaga aataacagga acgagggcac cctggatagc 1620 agaaggacca caaccaggca ctccaagaca atggaggagc tgaagctgcc tgagcacatg 1680 gactctagcc actcccactc tctgagcgcc ccacacgagt ccttctctta cggcctgggc 1740 tataccagcc ctttttcctc tcaacaacgc ccacaccggc actccatgta cgtgacacgg 1800 gacaaggtga gagccaaggg cctggacgg agcctgtcca tcggacaggg aatggcagca 1860 agggcaaata gcctgcagct gctgtcccca cagcctggag agcagctgcc accagagatg 1920 accgtggcac ggagctccgt gaaggaca agcagagagg gcaccttag cttccacaca 1980 cggcaaga gcgagggcgg cgtgtatcac gacccacact ccgacgacgg aaccgcacct 2040 aaggaaca gacacctgta caacgatcca gtgccccgcc gggtgggctc tttctatcgg 2100 gtgcctagcc cacgccccga caactccttt cacgagataa acgtgtctac ccgggtgtcc 2160 tctctgccca gcgagagctc ctctggcaca aaccactcca agagacagcc agcctttgat 2220 2280 ggcttctttc ggtccatgaa gaagagaag aagagagcc agacaaccga cagcaccaac 2340 ggcgagaatc caagcatcaa gaagtccctg ttccccctgt ttaacagcaa gaatcacctg 2400 aagcacagct cctctctgaa gaagctgccc gtggtgacac ctccaatggt gcccaattcc 2460 gactctcctg atctgctgac cctgcagaag tccatccaca gcgcctccac accaagctcc 2520 cggcccaagg agtggcggcc cgagaagatc agcgatctgc agacccagtc ccagcccctg 2580 aagtctctga gaaagctgct gcacctgtct agcgcctcca accacccagc ctcctctgac 2640 cctcggttcc agccactgac agcccagcag accaagaact cttttagcga gatccgcatc 2700 cacccactgt cccaagcctc tggaggcagc tccaacatca ggcaggagcc tgccccaaag 2760 ggccgcccag ccctgcagct gccaggacag atggaccccg gctggcacgt gtctagcgtg 2820 acacggagcg ccaccgaggg accttcctac tctgagcagc tgggagcaaa gtctggacca 2880 aacggacacc cttataacag aaccaataga agcaggatgc caaatctgaa cgacctgaag 2940 gagacagccc tgtgatag 2958 <210> 27 <211> 588 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence <400> 27 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 60 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 120 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 180 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 240 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 300 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 360 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 420 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 480 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 540 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgca 588 <210> 28 <211> 239 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence <400> 28 ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa 60 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca 120 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc 180 aggggggagat gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatc 239 <210> 29 <211> 7627 <212> DNA <213> Artificial sequence <220> <223> Expression Box <220> <221> repeat_region <222> (1)..(130) <223> 5' ITR <220> <221> misc_feature <222> (130) (168) <223> AAV2 sequence upstream of 3' ITR <220> <221> promoter <222> (226)..(1454) <223> UBC Starter <220> <221> misc_feature <222> (308)..(308) <223> Insert (C) <220> <221> misc_feature <222> (1009)..(1009) <223> Insert (G) <220> <221> CDS <222> (1470)...(4352) <223> hCDKL5_1 <220> <221> misc_feature <222> (4353)..(4353) <223> Missing (G) <220> <221> misc_feature <222> (4370)...(4391) <223> miRNA183 <220> <221> misc_feature <222> (4396) (4417) <223> miRNA183 <220> <221> misc_feature <222> (4424)...(4445) <223> miRNA183 <220> <221> misc_feature <222> (4452)...(4473) <223> miRNA183 <220> <221> polyA_signal <222> (4482)..(4608) <223> Rabbit globin polyA <220> <221> misc_feature <222> (4639)...(4677) <223> AAV2 sequence upstream of 3' ITR <220> <221> repeat_region <222> (4677)...(4806) <223> 3'ITR <400> 29 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180 aggaagatcc tctagaacta tagctagcat gctctaggaa gatctggcct ccgcgccggg 240 ttttggcgcc tcccgcgggc gcccccctcc tcacggcgag cgctgccacg tcagacgaag 300 ggcgcagcga gcgtcctgat ccttccgccc ggacgctcag gacagcggcc cgctgctcat 360 aagactcggc cttagaaccc cagtatcagc agaaggacat tttaggacgg gacttgggtg 420 actctagggc actggttttc tttccagaga gcggaacagg cgaggaaaag tagtcccttc 480 tcggcgattc tgcggaggga tctccgtggg gcggtgaacg ccgatgatta tataaggacg 540 cgccgggtgt ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg ttcttgtttg 600 tggatcgctg tgatcgtcac ttggtgagta gcgggctgct gggctggccg gggctttcgt 660 ggccgccggg ccgctcggtg ggacggaagc gtgtggagag accgccaagg gctgtagtct 720 gggtccgcga gcaaggttgc cctgaactgg gggttggggg gagcgcagca aaatggcggc 780 tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg ttgaaacaag 840 gtggggggca tggtgggcgg caagaaccca aggtcttgag gccttcgcta atgcgggaaa 900 gctcttattc gggtgagatg ggctggggca ccatctgggg accctgacgt gaagtttgtc 960 actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt 1020 gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt 1080 ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta ggcttttctc 1140 cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca ggcgccggac 1200 ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt atgtacctat 1260 cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc gagtgtgttt 1320 tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg taattttcag 1380 tgttagacta gtaaattgtc cgctaaattc tggccgtttt tggctttttt gttagacgaa 1440 gctttattgc ggtagaattc gccgccacc atg aag atc ccc aac atc ggc aac 1493 gtg atg aac aag ttc gag atc ctg ggc gtt gtc gga gaa ggc gct tat 1541 ggc gtg gtg ctg aag tgc aga cac aaa gaa acc cac gag atc gtg gcc 1589 atc aag aag ttc aag gac agc gaa gag aac gag gaa gtg aaa gag aca 1637 acc ctg cgc gag ctg aag atg ctg aga acc ctg aag caa gag aac atc 1685 gtg gaa ctg aaa gag gcc ttc cgg cgg aga ggc aag ctg tac ctg gtg 1733 ttc gag tac gtg gaa aag aac andg ctc gag ctg ctg gaa gag andg ccc 1781 aat ggc gtg cca cct gag aaa gtg aag tcc tac atc tac cag ctg atc 1829 aag gcc etc cac tgg tgc cac aag aac gac etc gtg cac aga gac etc 1877 aag ccc gag aac ctg ctg atc tcc cac aac gac gtg ctg aag ctg tgc 1925 gac ttc ggc ttc gcc aga aat ctg agc gag ggc aac aac gcc aac tac 1973 acc gag tat gtg gcc acc aga tgg tac aga agc cct gaa ctg ctg ctg 2021 gga gcc cct tac ggc aag tct gtg gat atg tgg tcc gtg ggc tgt atc 2069 ctg gga gag ctg tct gat ggc caa cct ctg ttt cct ggc gag tcc gag 2117 atc gac cag ctg ttc aca atc ​​cag aag gtg ctg ggc cct ctg cct agc 2165 gag cag atg aag ctg ttc tac agc aac ccc aga ttc cac ggc ctg aga 2213 ttc cct gcc gtg aat cac cct cag agc ctg gaa aga aga tac ctg ggc 2261 atc ctg aac agc gtg ctg ctg gac ctg atg aag aat ctg ctg aaa ctg 2309 gac ccc gcc gac aga tac ctg acc gag cag tgt ctg aac cat cct acc 2357 ttt cag acc cag aga ctg ctg gat aga agc ccc agc aga agc gcc aag 2405 aga aag ccc tac cac gtg gaa agc agc acc ctg agc aat aga aac cag 2453 gcc gga aag agc act gcc ctg cag agc cac cac aga agc aac agc aag 2501 gac atc cag aac ctg agc gtg ggc ctg cct aga gct gat gaa gga ctg 2549 cct gcc aac gag agc ttc ctg aat gga aat ctg gct ggc gcc tct ctg 2597 agc cct ctg cac aca aag aca tac cag gcc agc agc cag cct ggc agc 2645 aca agc aag gac ctg acc aac aac aac atc cct cat ctg ctg agc ccc 2693 aaa gag gcc aag agc aag acc gag ttc gac ttc aac atc gac ccc aag 2741 cct agc gaa ggc cct ggc acc aag tac ctg aag tcc aac tcc aga agc 2789 cag cag aac cgg cac tcc ttc atg gaa agc tct cag agc aag gcc ggc 2837 aca ctg cag cct aac gag aag cag agc aga cac agc tac atc gac aca 2885 atc cct cag tcc tct cgg agc ccc agc tac aga aca aag gcc aaa tct 2933 cac ggc gcc ctg agc gat agc aag agc gtg tcc aat ctg tcc gag gct 2981 aga gcc cag atc gcc gag cct agc acc agc aga tac ttc cct agc agc 3029 tgc ctg gat ctg aac agc cct aca agc ccc aca cct acc agg cac agc 3077 gat acc aga aca ctg ctg tcc cca agc ggc cgg aac aac aga aat gag 3125 ggc acc ctg gac agc aga cgg acc acc aca aga cac agc aag acc atg 3173 gaa gaa ctg aag ctg ccc gag cac atg gac tct agc cac tct cat tct 3221 ctg agc gcc cct cac gag tcc ttt agc tac ggc ctg ggc tac aca agc 3269 cca ttt tcc agt cag cag cgg ccc cac aga cac tct atg tac gtg acc 3317 aga gac aaa gtc cgc gcc aaa ggc ctg gat ggc tct ctg tct att gga 3365 caa ggc atg gcc gcc aga gcc aat agc ctg caa ctt ctg tct cct cag 3413 cct ggc gaa cag ctg cct cct gaa atg aca gtg gcc cgg tcc agc gtg 3461 aaa gaa acc agc aga gag ggc acc tcc agc ttc cac acc aga cag aaa 3509 agc gaa ggc ggc gtg tac cac gat cct cac tct gat gat ggc aca gcc 3557 cct aaa gag aac aga cac ctg tac aac gac ccc gtg cct aga aga gtg 3605 ggc tcc ttc tac aga gtg cct tct cct aga cct gac aac tcc ttc cac 3653 gag aac aac gtg tca acc cgg gtg tcc agc ctg cct tct gag agc agc 3701 tct ggc aca aac cac tcc aag agg cag ccc gcc ttc gat cct tgg aag 3749 tcc cct gag aat atc agc cac tcc gag cag ctg aaa gag aaa gag aag 3797 cag ggc ttt ttc cgg tcc atg aag aag aag aaa aag aag tcc cag aca 3845 gtc ccc aac agc gac agc cct gat ctc ctg aca ctg cag aag tcc atc 3893 cac agc gcc agc aca cct agc agc aga cct aaa gag tgg cgg cca gag 3941 aag atc agc gac ctg caa aca cag agc cag cct ctg aag tcc ctg agg 3989 aag ctg ctg cat ctg tcc tcc gcc tct aat cac cct gcc agc agc gat 4037 ccc aga ttt cag cca ctg aca gcc cag cag acc aag aac tcc ttc agc 4085 gag atc cgg att cac cca ctg tct cag gcc agc ggc ggc agc tct aat 4133 atc aga caa gag ccc gct cct aag ggc aga cct gct ctc caa ctg cct 4181 gga cag atg gat cct ggc tgg cac gtg tcc tct gtg aca aga tct gcc 4229 acc gag gga cct agc tat tct gag cag ctg ggc gcc aag tct ggc ccc 4277 aat gga cac ccc tac aac cgg acc aat cgg agc aga atg ccc aac ctg 4325 aac gac ctc aaa gaa aca gcc ctc tga agcttatcga taccggtagt 4372 gaattctacc agtgccatag gatagtgaat tctaccagtg catacacgt gagtgaattc 4432 taccagtgcc atagcatgca gtgaattcta ccagtgccat agcggccgcg atctttttcc 4492 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata 4552 aaggaaattt atttcattg caatagtgtg ttggaattttt ttgtgtctct cactcgatcg 4612 ataaggatct tcctagagca tggctacgta gataagtagc atggcgggtt aatcattaac 4672 tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact 4732 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccggcggc ctcagtgagc 4792 gagcgagcgc gcagccttaa ttaacctaat tcactggccg tcgttttaca acgtcgtgac 4852 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc ttcgccagc 4912 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 4972 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 5032 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 5092 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 5152 ttccgattta gtgctttacg gcacctcgac ccccaaaaaac ttg...

Claims

1. A recombinant adeno-associated virus (rAAV), wherein the rAAV comprises: (a) Clade F AAV capsid; as well as (b) A vector genome packaged in the AAV capsid of (a), wherein the vector genome comprises an inverted terminal repeat (ITR) sequence and an hCDKL5 coding sequence, the hCDKL5 coding sequence being a nucleic acid sequence encoding a functional human CDKL5 (hCDKL5), the nucleic acid sequence being under the control of a regulatory sequence regulating the expression of the hCDKL5, and wherein the hCDKL5 coding sequence is SEQ ID NO: 22 or at least 99% identical to SEQ ID NO: 22, and wherein the functional hCDKL5 has the amino acid sequence of SEQ ID NO: 2, and wherein the regulatory sequence comprises a human synaptic protein (hSyn) promoter or a ubiquitin C (UbC) promoter, and The regulatory sequence further includes at least two dorsal root ganglion (drg) specific miRNA-183 target sequences.

2. The rAAV according to claim 1, wherein the regulatory sequence further comprises one or more of a Kozak sequence, an intron, an enhancer, a TATA signal, and a polyA sequence.

3. The rAAV according to claim 1 or 2, wherein the regulatory sequence further comprises a rabbit β-globin polyadenylation (polyA) sequence.

4. The rAAV according to claim 1, wherein the miRNA-183 target sequence is SEQ ID NO:

11.

5. The rAAV according to claim 1 or 2, wherein two or more miRNA target sequences in the miRNA-183 target sequence are separated by spacers, and each spacer is independently selected from one or more of the following: (A) GGAT; (B) CACGTG; or (C) GCATGC.

6. The rAAV of claim 5, wherein the spacer located between the miRNA target sequences is located at the 3' of the first miRNA target sequence and / or the 5' of the last miRNA target sequence.

7. The rAAV of claim 5, wherein the spacers between the miRNA target sequences are identical.

8. The rAAV according to any one of claims 1 to 2, wherein the capping is an AAVhu68 capping or an AAV9 capping.

9. A composition comprising a stock solution of rAAV according to any one of claims 1 to 8 and an aqueous suspension medium.

10. The composition of claim 9, wherein the suspension is formulated for intravenous administration, intrathecal administration, intracerebellomedullary cistern administration, or intraventricular administration.

11. Use of the rAAV according to any one of claims 1 to 8 or the composition according to claim 9 or 10 in the preparation of a medicament for treating CDKL5 deficiency (CDD).

12. An rAAV generation system for generating rAAV according to any one of claims 1 to 7, wherein the generation system comprises a cell culture, the cell culture comprising: (a) The nucleic acid sequence encoding the capsid protein of clade F; (b) Vector genome; and (c) Sufficient to allow the AAV rep function and auxiliary functions of packaging the vector genome into the capsid of the clade F; The vector genome includes SEQ ID NO:

29.

13. The rAAV generation system according to claim 12, wherein the cell culture is a human embryonic kidney 293 cell culture.

Images